Formulations of Human Growth Hormone Comprising a Non-Naturally Encoded Amino Acid

ABSTRACT

Formulations of modified human growth hormone polypeptides are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional patent applicationSer. No. 60/638,616 filed Dec. 22, 2004, U.S. provisional patentapplication Ser. No. 60/680,617, filed May 13, 2005, and U.S.provisional patent application entitled 60/728,035, filed Oct. 17, 2005,the specifications of which are incorporated herein in their entirety.

FIELD OF THE INVENTION

This invention relates to stabilized human growth hormone (hGH)formulations of hGH polypeptide comprising a non-natural amino acidcovalently linked to poly(ethylene glycol) (PEG).

BACKGROUND OF THE INVENTION

Human growth hormone participates in much of the regulation of normalhuman growth and development. This naturally-occurring single-chainpituitary hormone consists of 191 amino acid residues and has amolecular weight of approximately 22 kDa. hGH exhibits a multitude ofbiological effects, including linear growth (somatogenesis), lactation,activation of macrophages, and insulin-like and diabetogenic effects,among others (Chawla, R., et al., Ann. Rev. Med. 34:519-547 (1983);Isaksson, O., et al., Ann. Rev. Physiol., 47:483-499 (1985); Hughes, J.and Friesen, H., Ann. Rev. Physiol., 47:469-482 (1985)).

The structure of hGH is well known (Goeddel, D., et al., Nature281:544-548 (1979)), and the three-dimensional structure of hGH has beensolved by X-ray crystallography (de Vos, A., et al., Science 255:306-312(1992)). The protein has a compact globular structure, comprising fouramphipathic alpha helical bundles, termed A-D beginning from theN-terminus, which are joined by loops. Further discussion of hGHincluding its receptor and variants, and other GH superfamily members isprovided in U.S. patent application Ser. No. 11/046,432 entitled“Modified Human Growth Hormone Polypeptides and Their Uses” and PCTInternational Patent Application No. PCT/US05/03537 entitled “ModifiedHuman Four Helical Bundle Polypeptides and Their Uses,” which areincorporated by reference in their entirety.

Recombinant hGH is used as a therapeutic and has been approved for thetreatment of a number of indications. hGH deficiency leads to dwarfism,for example, which has been successfully treated for more than a decadeby exogenous administration of the hormone. In addition to hGHdeficiency, hGH has also been approved for the treatment of renalfailure (in children), Turner's Syndrome, and cachexia in AIDS patients.Recently, the Food and Drug Administration (FDA) has approved hGH forthe treatment of non-GH-dependent short stature. hGH is also currentlyunder investigation for the treatment of aging, frailty in the elderly,short bowel syndrome, and congestive heart failure. Target populationsfor hGH treatment include children with idiopathic short stature (ISS)and adults with GHD-like symptoms.

Recombinant hGH is currently sold as a daily injectable product, withfive major products currently on the market: Humatrope™ (Eli Lilly &Co.), Nutropin™ (Genentech), Norditropin™ (Novo-Nordisk), Genotropin™(Pfizer) and Saizen/Serostim™ (Serono). A significant challenge to usinggrowth hormone as a therapeutic, however, is that the protein has ashort in vivo half-life and, therefore, it must be administered by dailysubcutaneous injection for maximum effectiveness (MacGillivray, et al.,J. Clin. Endocrinol. Metab. 81: 1806-1809 (1996)). Considerable effortis focused on means to improve the administration of hGH agonists andantagonists, by lowering the cost of production, making administrationeasier for the patient, improving efficacy and safety profile, andcreating other properties that would provide competitive advantages. Forexample, Genentech and Alkermes formerly marketed Nutropin Depot™, adepot formulation of hGH, for pediatric growth hormone deficiency. Whilethe depot permits less frequent administration (once every 2-3 weeksrather than once daily), it is also associated with undesirable sideeffects, such as decreased bioavailability and pain at the injectionsite and was withdrawn from the market in 2004. Another product,Pegvisomant™ (Pfizer), has also recently been approved by the FDA.Pegvisomant™ is a genetically-engineered analogue of hGH that functionsas a highly selective growth hormone receptor antagonist indicated forthe treatment of acromegaly (van der Lely, et al., The Lancet 358:1754-1759 (2001). Although several of the amino acid side chain residuesin Pegvisomant™ are derivatized with polyethylene glycol (PEG) polymers,the product is still administered once-daily, indicating that thepharmaceutical properties are not optimal. In addition to PEGylation anddepot formulations, other administration routes, including inhaled andoral dosage forms of hGH, are under early-stage pre-clinical andclinical development and none have yet received approval from the FDA.Accordingly, there is a need for a polypeptide that exhibits growthhormone activity but that also provides a longer serum half-life and,therefore, more optimal therapeutic levels of hGH and an increasedtherapeutic half-life.

Covalent attachment of the hydrophilic polymer poly(ethylene glycol),abbreviated PEG, is a method of increasing water solubility,bioavailability, increasing serum half-life, increasing therapeutichalf-life, modulating immunogenicity, modulating biological activity, orextending the circulation time of many biologically active molecules,including proteins, peptides, and particularly hydrophobic molecules.PEG has been used extensively in pharmaceuticals, on artificialimplants, and in other applications where biocompatibility, lack oftoxicity, and lack of immunogenicity are of importance. In order tomaximize the desired properties of PEG, the total molecular weight andhydration state of the PEG polymer or polymers attached to thebiologically active molecule must be sufficiently high to impart theadvantageous characteristics typically associated with PEG polymerattachment, such as increased water solubility and circulating halflife, while not adversely impacting the bioactivity of the parentmolecule.

Recently, an entirely new technology in the protein sciences has beenreported, which promises to overcome many of the limitations associatedwith site-specific modifications of proteins. Specifically, newcomponents have been added to the protein biosynthetic machinery of theprokaryote Escherichia coli (E. coli) (e.g., L. Wang, et al., (2001),Science 292:498-500) and the eukaryote Sacchromyces cerevisiae (S.cerevisiae) (e.g., J. Chin et al., Science 301:964-7 (2003)), which hasenabled the incorporation of non-genetically encoded amino acids toproteins in vivo. A number of new amino acids with novel chemical,physical or biological properties, including photoaffinity labels andphotoisomerizable amino acids, photocrosslinking amino acids (see, e.g.,Chin, J. W., et al. (2002) Proc. Natl. Acad. Sci. U.S.A. 99:11020-11024;and, Chin, J. W., et al., (2002) J. Am. Chem. Soc. 124:9026-9027), ketoamino acids, heavy atom containing amino acids, and glycosylated aminoacids have been incorporated efficiently and with high fidelity intoproteins in E. coli and in yeast in response to the amber codon, TAG,using this methodology. See, e.g., J. W. Chin et al., (2002), Journal ofthe American Chemical Society 124:9026-9027; J. W. Chin, & P. G.Schultz, (2002), Chem Bio Chem 3(11):1135-1137; J. W. Chin, et al.,(2002), PNAS United States of America 99:11020-11024; and, L. Wang, & P.G. Schultz, (2002), Chem. Comm., 1:1-11. All references are incorporatedby reference in their entirety. These studies have demonstrated that itis possible to selectively and routinely introduce chemical functionalgroups, such as ketone groups, alkyne groups and azide moieties, thatare not found in proteins, that are chemically inert to all of thefunctional groups found in the 20 common, genetically-encoded aminoacids and that may be used to react efficiently and selectively to formstable covalent linkages.

The ability to incorporate non-genetically encoded amino acids intoproteins permits the introduction of chemical functional groups thatcould provide valuable alternatives to the naturally-occurringfunctional groups, such as the epsilon —NH₂ of lysine, the sulfhydryl—SH of cysteine, the imino group of histidine, etc.

Human growth hormone formulations may be lyophilized preparationsrequiring reconstitution or aqueous formulations. Per vial, Protropin™hGH consists of 5 mg hGH, 40 mg mannitol, 0.1 mg monobasic sodiumphosphate, 1.6 mg dibasic sodium phosphate, reconstituted to pH 7.8(Physician's Desk Reference, Medical Economics Co., Orawell, N.J., p.1049, 1992). Per vial, Humatrope™ hGH consists of 5 mg hGH, 25 mgmannitol, 5 mg glycine, 1.13 mg dibasic sodium phosphate, reconstitutedto pH 7.5 (Physician's Desk Reference, p. 1266, 1992). Examples ofaqueous human growth hormone formulations are described in U.S. Pat.Nos. 5,763,394; 5,981,485; 6,448,225; and U.S. Patent Publication No.2003/0013653, each of which are incorporated by reference herein.

For a general review for growth hormone formulations, see Pearlman etal., Current Communications in Molecular Biology, eds. D. Marshak and D.Liu, pp. 23-30, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989, which is incorporated by reference herein. Otherpublications of interest regarding stabilization of proteins are asfollows.

U.S. Pat. No. 4,297,344, which is incorporated by reference herein,discloses stabilization of coagulation factors II and VIII, antithrombinIII, and plasminogen against heat by adding selected amino acids such asglycine, alanine, hydroxyproline, glutamine, and aminobutyric acid, anda carbohydrate such as a monosaccharide, an oligosaccharide, or a sugaralcohol.

U.S. Pat. No. 4,783,441, which is incorporated by reference herein,discloses a method for the prevention of denaturation of proteins suchas insulin in aqueous solution at interfaces by the addition of up to500 ppm surface-active substances comprising a chain of alternating,weakly hydrophilic and weakly hydrophobic zones at pH 6.8-8.0.

U.S. Pat. No. 4,812,557, which is incorporated by reference herein,discloses a method of stabilization of interleukin-2 using human serumalbumin.

European Patent Application Publication No. 0 303 746, which isincorporated by reference herein, discloses stabilization of growthpromoting hormones with polyols consisting of non-reducing sugars, sugaralcohols, sugar acids, pentaerythritol, lactose, water-soluble dextrans,and Ficoll, amino acids, polymers of amino acids having a charged sidegroup at physiological pH, and choline salts.

European Patent Application Publication No. 0 211 601, which isincorporated by reference herein, discloses the stabilization of growthpromoting hormones in a gel matrix formed by a block copolymercontaining polyoxyethylene-polyoxypropylene units and having an averagemolecular weight of about 1,100 to about 40,000.

European Patent Application Publication No. 0 193 917, which isincorporated by reference herein, discloses a biologically activecomposition for slow release characterized by a water solution of acomplex between a protein and a carbohydrate.

International Patent Publication No. WO 89/09614 and Australian patentapplication No. 30771/89, which are incorporated by reference herein,disclose a stable pharmaceutical formulation containing human growthhormone, glycine, and mannitol. Such a preparation shows improvedstability during normal processing and storage in a lyophilized state aswell as in the period of use after the reconstitution.

U.S. Pat. No. 5,096,885, which is incorporated by reference herein,discloses a formulation of hGH for lyophilization containing glycine,mannitol, a non-ionic surfactant, and a buffer.

U.S. Pat. No. 4,876,568, which is incorporated by reference herein,discloses that animal growth hormone may be stabilized with variousstabilizers to give decreased formation of insolubles and preservationof the soluble activity in aqueous environments. Such stabilizersincluding certain polyols, amino acids, polymers of amino acids having acharged side group at physiological pH, and choline salts. Polyols areselected from the group consisting of non-reducing sugars, sugaralcohols, sugar acids, pentaerythritol, lactose, water-soluble dextransand Ficoll; amino acids are selected from the group consisting ofglycine, sarcosine, lysine or salts thereof, serine, arginine or saltsthereof, betaine, N,N,-dimethyl-glycine, aspartic acid or salts thereof,glutamic acid or salts thereof; a polymer of an amino acid having acharged side group at physiological pH may be selected from polylysine,polyaspartic acid, polyglutamic acid, polyarginine, polyhistidine,polyornithine and salts thereof; and choline derivatives are selectedfrom the group consisting of choline chloride, choline dihydrogencitrate, choline bitartrate, choline bicarbonate, tricholine citrate,choline ascorbate, choline borate, choline gluconate, choline phosphate,di(choline)sulphate and dicholine mucate. U.S. Pat. No. 4,876,568, whichis incorporated by reference herein, notes that polyhistidine can beused as a potential stabilizer for animal growth hormone but there is noindication whether it stabilizes an animal growth hormone or humangrowth hormone. Furthermore, U.S. Pat. No. 4,876,568 mentions thatpoly-DL-lysine HBr is preferred.

EP 374120, which is incorporated by reference herein, discloses astabilized preparation of growth hormone comprising a buffered polyolexcipient comprising a polyol having three hydroxy groups and a bufferto achieve a pH in a range in which the growth hormone retains itsbioactivity for a sufficient period of time. Histidine is mentioned as abuffer for a polyol having three hydroxy groups. Specifically, EP 374120teaches that histidine hydrochloride may be used as a buffer forbuffering a polyol having three hydroxy groups for improving thestability of a growth hormone preparation in the form of a solutioncomprising a high concentration of growth hormone and a polyol asstabilizer. Furthermore, histidine hydrochloride must be added in anamount of about 3% by weight of the solution corresponding to aconcentration of ˜0.15M solution of histidine hydrochloride. EP 374120also teaches that histidine alone does not impart chemical and physicalstability to a growth hormone preparation.

Sorensen et al., WO 93/12812, which is incorporated by reference herein,teaches that growth hormone can be stabilized by the presence ofhistidine or a histidine derivative. If the growth hormone islyophilized, the composition can also comprise a bulking agent, i.e.sugar alcohols, disaccharides, and mixtures thereof. Sorensen et al.,U.S. Pat. No. 5,849,704, which is incorporated by reference herein,discloses a pharmaceutical formulation comprising a growth hormone andhistidine or a derivative of histidine as an additive or bufferingsubstance added to provide stability against deamidation, oxidation orcleavage of the peptide bonds in the growth hormone. Also disclosed isthat crystallization of growth hormone in the presence of histidine or aderivative thereof gives rise to a higher yield of crystals havinghigher purity than known methods. Formulations of human growth hormonevariants have been described in U.S. Pat. Nos. 6,136,563 and 5,849,535,which are incorporated by reference herein.

hGH undergoes breakdown via several degradative pathways, includingdeamidation, aggregation, clipping of the peptide backbone, andoxidation of methionine residues. Additional products result fromdegradation of conjugates of hGH covalently attached to a water solublepolymer such PEG. A pharmaceutical formulation of hGH that providesacceptable control of degradation products, has maintained stability ofhGH over a prolonged period of time, and is stable to vigorous agitation(which induces aggregation) would be particularly advantageous.

BRIEF SUMMARY OF THE INVENTION

This invention provides formulations of hGH polypeptides comprising oneor more non-naturally encoded amino acids.

In some embodiments, the hGH polypeptide comprises one or morepost-translational modifications. In some embodiments, the hGHpolypeptide is linked to a linker, polymer, or biologically activemolecule. In some embodiments, the hGH polypeptide is linked to abifunctional polymer, bifunctional linker, or at least one additionalhGH polypeptide.

In some embodiments, the non-naturally encoded amino acid is linked to awater soluble polymer. In some embodiments, the water soluble polymercomprises a poly(ethylene glycol) moiety. In some embodiments, thenon-naturally encoded amino acid is linked to the water soluble polymerwith a linker or bonded to a water soluble polymer. In some embodiments,the water soluble polymer comprises a poly(ethylene glycol) moiety. Insome embodiments, the invention is a single-dose lyophilized formulationof hGH polypeptide. In some embodiments, the invention is a liquidformulation of hGH polypeptide.

In some embodiments, the poly(ethylene glycol) molecule has a molecularweight of between about 0.1 kDa and about 100 kDa. In some embodiments,the poly(ethylene glycol) molecule has a molecular weight of between 0.1kDa and 50 kDa.

In some embodiments, the poly(ethylene glycol) molecule is a branchedpolymer. In some embodiments, each branch of the poly(ethylene glycol)branched polymer has a molecular weight of between 1 kDa and 100 kDa, orbetween 1 kDa and 50 kDa.

In one embodiment, the pharmaceutical formulation of hGH polypeptidecomprising one or more non-naturally encoded amino acids comprises abuffer, at least one a carrier, excipient, or stabilizer, and apharmaceutical quantity of human growth hormone (hGH). In anotherembodiment, the at least one a carrier, excipient, or stabilizer isselected from the group consisting of an antioxidant, an amino acid, acarbohydrate, a chelating agent, a sugar alcohol, a salt-forming counterion, and a non-ionic surfactant. The present invention also providesmethods of treating a patient having a disorder modulated by hGH with aneffective amount of the formulation of a hGH molecule of the presentinvention.

In one embodiment of the present invention, formulations of hGHpolypeptide comprising a non-naturally encoded amino acid that minimizeformation of undesirable aggregated species or cause chemical changesthat reduce biological activity or alter receptor recognition areprovided. Such formulations are capable of maintaining activity forappropriate storage times, are readily formulated, and are acceptablefor administration to patients.

In one embodiment, the formulation of hGH polypeptide, including but notlimited to PEGylated hGH, comprising one or more non-naturally encodedamino acids is a lyophilized formulation that is reconstituted prior touse for subcutaneous injection. The formulations of the presentinvention may be pharmaceutical formulations, in particular,formulations for subcutaneous administration.

In another embodiment, the invention provides a method for thetreatment, prophylactic or therapeutic, of a disorder treatable by theprotein formulated, including but not limited to, PEGylated hGH, usingthe formulations disclosed herein. Such formulations are particularlyuseful for subcutaneous administration.

Also provided is an article of manufacture comprising a containerenclosing a formulation disclosed herein, as well as pre-filledsyringes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the pH stability analysis of formulation buffers after sixweeks at 4° C.

FIG. 2 shows SDS-PAGE reducing (FIGS. 2C and 2D) and non-reducing gels(FIGS. 2A and 2B) of Met Y35pAF hGH. Gels in FIGS. 2A and 2B are loadedas follows: Lane 1: MW, Lane 2: WHO hGH, Lane 3: WHO hGH 1%; Lane 4: A1;Lane 5: A2; Lane 6: A3; Lane 7: A4; Lane 8: A5; Lane 9: A6; Lane 10: A7;Lane 11: A8; Lane 12: MW. Gels in FIGS. 2C and 2D are loaded as follows:Lane 1: MW, Lane 2: WHO hGH, Lane 3: WHO hGH 1%; Lane 4: B1; Lane 5: B2;Lane 6: B3; Lane 7: B4; Lane 8: B5; Lane 9: B6; Lane 10: B7; Lane 11:B8; Lane 12: MW.

FIG. 3A-F shows Differential Scanning Calorimetry (DSC) thermoprofilesof formulation groups A-F.

FIG. 4 shows DSC thermoprofiles from groups B7 (FIG. 4A) and F2 (FIG.4B) of the matrix.

FIG. 5 provides a table summarizing the DSC melting temperatures andchanges to T_(m) for the full matrix.

FIG. 6 shows a summary of the DSC melting temperatures for the fullmatrix.

FIG. 7 shows an analysis of the ratio of ΔH_(v)/ΔH for the formulationgroups.

FIG. 8 shows an analysis of the change in enthalpy (AUC) of each sample.

FIG. 9A-D shows RP-HPLC datasets of samples in Group B, C, E, and Fcompared to the WHO hGH standard.

FIG. 10A shows an analysis of the different Y35pAF peaks for Group E5(Relative percent of all peaks were plotted at each time point tovisualize any and all changes to the main MetY35pAF hGH peak), and FIG.10B shows a zoom of the primary deamination/oxidation peak (Zoomed inDeamidation Peak—increase in deamidation over time (t=0 to 4 weeks).RP-HPLC Analysis was performed using Agilent Chemstation Software.

FIG. 11 shows an analysis of the primary deamidation/oxidation peakacross the matrix. (Analysis of the Met Y35pAF hGH Over 4 weeks@4° C.)

FIG. 12 shows an analysis of the secondary deamidation/oxidation peakwith the formulation groups. (Analysis of the Met Y35pAF hGH Over 4weeks@4° C.)

FIG. 13 shows an analysis of the main GH peak for the formulationgroups. (Analysis of the Met Y35pAF hGH Over 4 weeks@ 4° C.)

FIG. 14 shows RP-HPLC analysis of the primary deamidation/oxidation peakin samples that had undergone freeze-thaw cycles with formulation groupsB and C. (Analysis of Met Y35pAF hGH Peak over 5 Freeze/Thaw Cycles)

FIG. 15 shows RP-HPLC analysis of the secondary deamidation peak insamples that had undergone freeze-thaw cycles with formulation groups Band C. (Analysis of Met Y35pAF hGH over 5 Freeze/Thaw Cycles)

FIG. 16 shows RP-HPLC analysis of the main GH peak in samples that hadundergone freeze-thaw cycles with formulation groups B and C. (Analysisof Met Y35pAF hGH over 5 Freeze/Thaw Cycles)

FIG. 17 shows results from an analysis by RP-HPLC.

FIG. 18 shows results from an analysis by SEC-HPLC.

FIG. 19 shows results from an analysis by cIEX-HPLC.

FIG. 20 shows an example of SEC-HPLC integration.

FIG. 21 shows an example of RP-HPLC integration.

FIG. 22 shows an SDS-PAGE analysis (reduced) of control samples in aformulation study (t=0). Lane 1: Mark 12; Lane 2: Ref. Std; Lane 3:P6MT; Lane 4: S4MT; Lane 5: S5MT; Lane 6: S5GT; Lane 7: H6MT; Lane 8:P6GT; Lane 9: P6MA; Lane 10: P6MS.

FIG. 23 shows an SDS-PAGE analysis (reduced) of control samples in aformulation study (t=0). Lane 1: Mark 12; Lane 2: Ref. Std; Lane 3:P6MTMet; Lane 4: P7MT; Lane 5: P7GT; Lane 6: P6MGT; Lane 7: P6MGT-P;Lane 8: P6MT-P; Lane 9: P6GT-P.

FIG. 24 shows an SDS-PAGE analysis (non-reduced) of control samples in aformulation study (t=0). For FIG. 24, panel A, Lane 1: Mark 12 Standard;Lane 2: PEG-hGH Standard; Lane 3: P6MT; Lane 4: S4MT; Lane 5: S5MT; Lane6: S5GT; Lane 7: H6MT; Lane 8: P6GT; Lane 9: P6MA; Lane 10: P6MS. ForFIG. 24, panel B, Lane 1: Mark 12 Standard; Lane 2: PEG-hGH Standard;Lane 3: P6MTMet; Lane 4: P7MT; Lane 5: P7GT; Lane 6: P6MGT; Lane 7:P6MGT-P; Lane 8: P6MT-P; Lane 9: P6GT-P.

FIG. 25 shows an SDS-PAGE analysis (non-reduced) of samples stored at 4°C. for 1 week. For FIG. 25, panel A, Lane 1: Mark 12 Standard; Lane 2:PEG-hGH Standard; Lane 3: P6MT; Lane 4: S4MT; Lane 5: S5MT; Lane 6:S5GT; Lane 7: H6MT; Lane 8: P6GT; Lane 9: P6MA; Lane 10: P6MS. For FIG.25, panel B, Lane 1: Mark 12 Standard; Lane 2: PEG-hGH Standard; Lane 3:P6MTMet; Lane 4: P7MT; Lane 5: P7GT; Lane 6: P6MGT; Lane 7: P6MGT-P;Lane 8: P6MT-P; Lane 9: P6GT-P.

FIG. 26 shows an SDS-PAGE analysis (reduced) of samples stored at 4° C.for 1 week. For FIG. 26, panel A, Lane 1: Mark 12 Standard; Lane 2:PEG-hGH Standard; Lane 3: P6MT; Lane 4: S4MT; Lane 5: S5MT; Lane 6:S5GT; Lane 7: H6MT; Lane 8: P6GT; Lane 9: P6MA; Lane 10: P6MS. For FIG.26, panel B, Lane 1: Mark 12 Standard; Lane 2: PEG-hGH Standard; Lane 3:P6MTMet; Lane 4: P7MT; Lane 5: P7GT; Lane 6: P6MGT; Lane 7: P6MGT-P;Lane 8: P6MT-P; Lane 9: P6GT-P.

FIG. 27 shows an SDS-PAGE analysis (non-reduced) of samples stored at 4°C. for 2 weeks. For FIG. 27, panel A, Lane 1: Mark 12 Standard; Lane 2:PEG-hGH Standard; Lane 3: P6MT; Lane 4: S4MT; Lane 5: S5MT; Lane 6:S5GT; Lane 7: H6MT; Lane 8: P6GT; Lane 9: P6MA; Lane 10: P6MS. For FIG.27, panel B, Lane 1: Mark 12 Standard; Lane 2: PEG-hGH Standard; Lane 3:P6MTMet; Lane 4: P7MT; Lane 5: P7GT; Lane 6: P6MGT; Lane 7: P6MGT-P;Lane 8: P6MT-P; Lane 9: P6GT-P.

FIG. 28 shows an SDS-PAGE analysis (reduced) of samples stored at 4° C.for 2 weeks. For FIG. 28, panel A, Lane 1: Mark 12 Standard; Lane 2:PEG-hGH Standard; Lane 3: P6MT; Lane 4: S4MT; Lane 5: S5MT; Lane 6:S5GT; Lane 7: H6MT; Lane 8: P6GT; Lane 9: P6MA; Lane 10: P6MS. For FIG.28, panel B, Lane 1: Mark 12 Standard; Lane 2: PEG-hGH Standard; Lane 3:P6MTMet; Lane 4: P7MT; Lane 5: P7GT; Lane 6: P6MGT; Lane 7: P6MGT-P;Lane 8: P6MT-P; Lane 9: P6GT-P.

FIG. 29 shows an SDS-PAGE analysis (non-reduced) of samples stored at 4°C. for 4 weeks. For FIG. 29, panel A, Lane 1: Mark 12 Standard; Lane 2:PEG-hGH Standard; Lane 3: P6MT; Lane 4: S4MT; Lane 5: S5MT; Lane 6:S5GT; Lane 7: H6MT; Lane 8: P6GT; Lane 9: P6MA; Lane 10: P6MS. For FIG.29, panel B, Lane 1: Mark 12 Standard; Lane 2: PEG-hGH Standard; Lane 3:P6MTMet; Lane 4: P7MT; Lane 5: P7GT; Lane 6: P6MGT; Lane 7: P6MGT-P;Lane 8: P6MT-P; Lane 9: P6GT-P.

FIG. 30 shows an SDS-PAGE analysis (reduced) of samples stored at 4° C.for 4 weeks. For FIG. 30, panel A, Lane 1: Mark 12 Standard; Lane 2:PEG-hGH Standard; Lane 3: P6MT; Lane 4: S4MT; Lane 5: S5MT; Lane 6:S5GT; Lane 7: H6MT; Lane 8: P6GT; Lane 9: P6MA; Lane 10: P6MS. For FIG.30, panel B, Lane 1: Mark 12 Standard; Lane 2: PEG-hGH Standard; Lane 3:P6MTMet; Lane 4: P7MT; Lane 5: P7GT; Lane 6: P6MGT; Lane 7: P6MGT-P;Lane 8: P6MT-P; Lane 9: P6GT-P.

FIG. 31 shows an SDS-PAGE analysis (non-reduced) of samples stored at 4°C. for 6 weeks. Lane 1: PEG-hGH Standard; Lane 3: P6MT; Lane 4: S5MT;Lane 5: S5GT; Lane 6: H6MT; Lane 7: P6GT; Lane 8: P6MS; Lane 9: P6MTMet;Lane 10: P6MT; Lane 11: P7GT; Lane 12: P6MGT; Lane 13: P6MGT-P; Lane 14:P6MT-P; Lane 15: P6GT-P.

FIG. 32 shows an SDS-PAGE analysis (reduced) of samples stored at 4° C.for 6 weeks. Lane 1: PEG-hGH Standard; Lane 3: P6MT; Lane 4: S5MT; Lane5: S5GT; Lane 6: H6MT; Lane 7: P6GT; Lane 8: P6MS; Lane 9: P6MTMet; Lane10: P6MT; Lane 11: P7GT; Lane 12: P6MGT; Lane 13: P6MGT-P; Lane 14:P6MT-P; Lane 15: P6GT-P.

FIG. 33 shows an SDS-PAGE analysis (non-reduced) of samples stored at 4°C. for 2 months. Lane 1: PEG-hGH Standard; Lane 3: hGH (1 μg); Lane 5:P6MT; Lane 6: H6MT; Lane 7: P6GT; Lane 8: P6MS; Lane 9: P6MTMet; Lane10: P6MT; Lane 11: P7GT; Lane 12: P6MGT; Lane 13: P6MGT-P; Lane 14:P6MT-P; Lane 15: P6GT-P.

FIG. 34 shows an SDS-PAGE analysis (reduced) of samples stored at 4° C.for 2 months. Lane 1: PEG-hGH Standard; Lane 3: hGH (1 μg); Lane 5:P6MT; Lane 6: H6MT; Lane 7: P6GT; Lane 8: P6MS; Lane 9: P6MTMet; Lane10: P6MT; Lane 11: P7GT; Lane 12: P6MGT; Lane 13: P6MGT-P; Lane 14:P6MT-P; Lane 15: P6GT-P.

FIG. 35 shows an SDS-PAGE analysis (non-reduced) of samples stored at 4°C. for 3 months. Lane 1: PEG-hGH Standard; Lane 3: hGH (1 μg); Lane 5:P6MT; Lane 6: H6MT; Lane 7: P6GT; Lane 8: P6MS; Lane 9: P6MTMet; Lane10: P6MT; Lane 11: P7GT; Lane 12: P6MGT; Lane 13: P6MGT-P; Lane 14:P6MT-P; Lane 15: P6GT-P.

FIG. 36 shows an SDS-PAGE analysis (reduced) of samples stored at 4° C.for 3 months. Lane 1: PEG-hGH Standard; Lane 3: hGH (1 μg); Lane 5:P6MT; Lane 6: H6MT; Lane 7: P6GT; Lane 8: P6MS; Lane 9: P6MTMet*(contamination); Lane 10: P6MT; Lane 11: P7GT; Lane 12: P6MGT; Lane 13:P6MGT-P; Lane 14: P6MT-P; Lane 15: P6GT-P.

FIG. 37 shows an SDS-PAGE analysis (non-reduced) of samples stored at25° C. for 1 week. For FIG. 37, panel A, Lane 1: Mark 12 Standard; Lane2: PEG-hGH Standard; Lane 3: P6MT; Lane 4: S4MT; Lane 5: S5MT; Lane 6:S5GT; Lane 7: H6MT; Lane 8: P6GT; Lane 9: P6MA; Lane 10: P6MS. For FIG.37, panel B, Lane 1: Mark 12 Standard; Lane 2: PEG-hGH Standard; Lane 3:P6MTMet; Lane 4: P7MT; Lane 5: P7GT; Lane 6: P6MGT; Lane 7: P6MGT-P;Lane 8: P6MT-P; Lane 9: P6GT-P.

FIG. 38 shows an SDS-PAGE analysis (reduced) of samples stored at 25° C.for 1 week. For FIG. 38, panel A, Lane 1: Mark 12 Standard; Lane 2:PEG-hGH Standard; Lane 3: P6MT; Lane 4: S4MT; Lane 5: S5MT; Lane 6:S5GT; Lane 7: H6MT; Lane 8: P6GT; Lane 9: P6MA; Lane 10: P6MS. For FIG.38, panel B, Lane 1: Mark 12 Standard; Lane 2: PEG-hGH Standard; Lane 3:P6MTMet; Lane 4: P7MT; Lane 5: P7GT; Lane 6: P6MGT; Lane 7: P6MGT-P;Lane 8: P6MT-P; Lane 9: P6GT-P.

FIG. 39 shows an SDS-PAGE analysis (non-reduced) of samples stored at25° C. for 2 weeks. For FIG. 39, panel A, Lane 1: Mark 12 Standard; Lane2: PEG-hGH Standard; Lane 3: P6MT; Lane 4: S4MT; Lane 5: S5MT; Lane 6:S5GT; Lane 7: H6MT; Lane 8: P6GT; Lane 9: P6MA; Lane 10: P6MS. For FIG.39, panel B, Lane 1: Mark 12 Standard; Lane 2: PEG-hGH Standard; Lane 3:P6MTMet; Lane 4: P7MT; Lane 5: P7GT; Lane 6: P6MGT; Lane 7: P6MGT-P;Lane 8: P6MT-P; Lane 9: P6GT-P.

FIG. 40 shows an SDS-PAGE analysis (reduced) of samples stored at 25° C.for 2 weeks. For FIG. 40, panel A, Lane 1: Mark 12 Standard; Lane 2:PEG-hGH Standard; Lane 3: P6MT; Lane 4: S4MT; Lane 5: S5MT; Lane 6:S5GT; Lane 7: H6MT; Lane 8: P6GT; Lane 9: P6MA; Lane 10: P6MS. For FIG.40, panel B, Lane 1: Mark 12 Standard; Lane 2: PEG-hGH Standard; Lane 3:P6MTMet; Lane 4: P7MT; Lane 5: P7GT; Lane 6: P6MGT; Lane 7: P6MGT-P;Lane 8: P6MT-P; Lane 9: P6GT-P.

FIG. 41 shows an SDS-PAGE analysis (non-reduced) of samples stored at25° C. for 4 weeks. For FIG. 41, panel A, Lane 1: Mark 12 Standard; Lane2: PEG-hGH Standard; Lane 3: P6MT; Lane 4: S4MT; Lane 5: S5MT; Lane 6:S5GT; Lane 7: H6MT; Lane 8: P6GT; Lane 9: P6MA; Lane 10: P6MS. For FIG.41, panel B, Lane 1: Mark 12 Standard; Lane 2: PEG-hGH Standard; Lane 3:P6MTMet; Lane 4: P7MT; Lane 5: P7GT; Lane 6: P6MGT; Lane 7: P6MGT-P;Lane 8: P6MT-P; Lane 9: P6GT-P.

FIG. 42 shows an SDS-PAGE analysis (reduced) of samples stored at 25° C.for 4 weeks. For FIG. 42, panel A, Lane 1: Mark 12 Standard; Lane 2:PEG-hGH Standard; Lane 3: P6MT; Lane 4: S4MT; Lane 5: S5MT; Lane 6:S5GT; Lane 7: H6MT; Lane 8: P6GT; Lane 9: P6MA; Lane 10: P6MS. For FIG.42, panel B, Lane 1: Mark 12 Standard; Lane 2: PEG-hGH Standard; Lane 3:P6MTMet; Lane 4: P7MT; Lane 5: P7GT; Lane 6: P6MGT; Lane 7: P6MGT-P;Lane 8: P6MT-P; Lane 9: P6GT-P.

FIG. 43 shows an SDS-PAGE analysis (non-reduced) of samples stored at25° C. for 6 weeks. Lane 1: PEG-hGH Standard; Lane 3: P6MT; Lane 4:S5MT; Lane 5: S5GT; Lane 6: H6MT; Lane 7: P6GT; Lane 8: P6MS; Lane 9:P6MTMet; Lane 10: P6MT; Lane 11: P7GT; Lane 12: P6MGT; Lane 13: P6MGT-P;Lane 14: P6MT-P; Lane 15: P6GT-P.

FIG. 44 shows an SDS-PAGE analysis (reduced) of samples stored at 25° C.for 6 weeks. Lane 1: PEG-hGH Standard; Lane 3: P6MT; Lane 4: S5MT; Lane5: S5GT; Lane 6: H6MT; Lane 7: P6GT; Lane 8: P6MS; Lane 9: P6MTMet; Lane10: P6MT; Lane 11: P7GT; Lane 12: P6MGT; Lane 13: P6MGT-P; Lane 14:P6MT-P; Lane 15: P6GT-P.

FIG. 45 shows an SDS-PAGE analysis (non-reduced) of samples stored at25° C. for 2 months. Lane 1: PEG-hGH Standard; Lane 3: hGH (1 μg); Lane5: P6MT; Lane 6: H6MT; Lane 7: P6GT; Lane 8: P6MS; Lane 9: P6MTMet; Lane10: P6MT; Lane 11: P7GT; Lane 12: P6MGT; Lane 13: P6MGT-P; Lane 14:P6MT-P; Lane 15: P6GT-P.

FIG. 46 shows an SDS-PAGE analysis (reduced) of samples stored at 25° C.for 2 months. Lane 1: PEG-hGH Standard; Lane 3: hGH (1 μg); Lane 5:P6MT; Lane 6: H6MT; Lane 7: P6GT; Lane 8: P6MS; Lane 9: P6MTMet; Lane10: P6MT; Lane 11: P7GT; Lane 12: P6MGT; Lane 13: P6MGT-P; Lane 14:P6MT-P; Lane 15: P6GT-P.

FIG. 47 shows an SDS-PAGE analysis (non-reduced) of samples stored at25° C. for 3 months. Lane 1: PEG-hGH Standard; Lane 3: hGH (1 μg); Lane5: P6MT; Lane 6: H6MT; Lane 7: P6GT; Lane 8: P6MS; Lane 9: P6MTMet; Lane10: P6MT; Lane 11: P7GT; Lane 12: P6MGT; Lane 13: P6MGT-P; Lane 14:P6MT-P; Lane 15: P6GT-P.

FIG. 48 shows an SDS-PAGE analysis (reduced) of samples stored at 25° C.for 3 months. Lane 1: PEG-hGH Standard; Lane 3: hGH (1 μg); Lane 5:P6MT; Lane 6: H6MT; Lane 7: P6GT; Lane 8: P6MS; Lane 9: P6MTMet; Lane10: P6MT; Lane 11: P7GT; Lane 12: P6MGT; Lane 13: P6MGT-P; Lane 14:P6MT-P; Lane 15: P6GT-P.

FIG. 49 shows an SDS-PAGE analysis (non-reduced) of samples stored at40° C. for 1 week. For FIG. 49, panel A, Lane 1: Mark 12 Standard; Lane2: PEG-hGH Standard; Lane 3: P6MT; Lane 4: S4MT; Lane 5: S5MT; Lane 6:S5GT; Lane 7: H6MT; Lane 8: P6GT; Lane 9: P6MA; Lane 10: P6MS. For FIG.49, panel B, Lane 1: Mark 12 Standard; Lane 2: PEG-hGH Standard; Lane 3:P6MTMet; Lane 4: P7MT; Lane 5: P7GT; Lane 6: P6MGT; Lane 7: P6MGT-P;Lane 8: P6MT-P; Lane 9: P6GT-P.

FIG. 50 shows an SDS-PAGE analysis (reduced) of samples stored at 40° C.for 1 week. For FIG. 50, panel A, Lane 1: Mark 12 Standard; Lane 2:PEG-hGH Standard; Lane 3: P6MT; Lane 4: S4MT; Lane 5: S5MT; Lane 6:S5GT; Lane 7: H6MT; Lane 8: P6GT; Lane 9: P6MA; Lane 10: P6MS. For FIG.50, panel B, Lane 1: Mark 12 Standard; Lane 2: PEG-hGH Standard; Lane 3:P6MTMet; Lane 4: P7MT; Lane 5: P7GT; Lane 6: P6MGT; Lane 7: P6MGT-P;Lane 8: P6MT-P; Lane 9: P6GT-P.

FIG. 51 shows an SDS-PAGE analysis (non-reduced) of samples stored at40° C. for 2 weeks. For FIG. 51, panel A, Lane 1: Mark 12 Standard; Lane2: PEG-hGH Standard; Lane 3: P6MT; Lane 4: S4MT; Lane 5: S5MT; Lane 6:S5GT; Lane 7: H6MT; Lane 8: P6GT; Lane 9: P6MA; Lane 10: P6MS. For FIG.51, panel B, Lane 1: Mark 12 Standard; Lane 2: PEG-hGH Standard; Lane 3:P6MTMet; Lane 4: P7MT; Lane 5: P7GT; Lane 6: P6MGT; Lane 7: P6MGT-P;Lane 8: P6MT-P; Lane 9: P6GT-P.

FIG. 52 shows an SDS-PAGE analysis (reduced) of samples stored at 40° C.for 2 weeks. For FIG. 52, panel A, Lane 1: Mark 12 Standard; Lane 2:PEG-hGH Standard; Lane 3: P6MT; Lane 4: S4MT; Lane 5: S5MT; Lane 6:S5GT; Lane 7: H6MT; Lane 8: P6GT; Lane 9: P6MA; Lane 10: P6MS. For FIG.52, panel B, Lane 1: Mark 12 Standard; Lane 2: PEG-hGH Standard; Lane 3:P6MTMet; Lane 4: P7MT; Lane 5: P7GT; Lane 6: P6MGT; Lane 7: P6MGT-P;Lane 8: P6MT-P; Lane 9: P6GT-P.

FIG. 53 shows an SDS-PAGE analysis (non-reduced) of samples stored at40° C. for 4 weeks. For FIG. 53, panel A, Lane 1: Mark 12 Standard; Lane2: PEG-hGH Standard; Lane 3: P6MT; Lane 4: S4MT; Lane 5: S5MT; Lane 6:S5GT; Lane 7: H6MT; Lane 8: P6GT; Lane 9: P6MA; Lane 10: P6MS. For FIG.53, panel B, Lane 1: Mark 12 Standard; Lane 2: PEG-hGH Standard; Lane 3:P6MTMet; Lane 4: P7MT; Lane 5: P7GT; Lane 6: P6MGT; Lane 7: P6MGT-P;Lane 8: P6MT-P; Lane 9: P6GT-P.

FIG. 54 shows an SDS-PAGE analysis (reduced) of samples stored at 40° C.for 4 weeks. For FIG. 54, panel A, Lane 1: Mark 12 Standard; Lane 2:PEG-hGH Standard; Lane 3: P6MT; Lane 4: S4MT; Lane 5: S5MT; Lane 6:S5GT; Lane 7: H6MT; Lane 8: P6GT; Lane 9: P6MA; Lane 10: P6MS. For FIG.54, panel B, Lane 1: Mark 12 Standard; Lane 2: PEG-hGH Standard; Lane 3:P6MTMet; Lane 4: P7MT; Lane 5: P7GT; Lane 6: P6MGT; Lane 7: P6MGT-P;Lane 8: P6MT-P; Lane 9: P6GT-P.

FIG. 55 shows an SDS-PAGE analysis (non-reduced) of samples stored at40° C. for 6 weeks. Lane 1: PEG-hGH Standard; Lane 3: P6MT; Lane 4:S5MT; Lane 5: S5GT; Lane 6: H6MT; Lane 7: P6GT; Lane 8: P6MS; Lane 9:P6MTMet; Lane 10: P6MT; Lane 11: P7GT; Lane 12: P6MGT; Lane 13: P6MGT-P;Lane 14: P6MT-P; Lane 15: P6GT-P.

FIG. 56 shows an SDS-PAGE analysis (reduced) of samples stored at 40° C.for 6 weeks. Lane 1: PEG-hGH Standard; Lane 3: P6MT; Lane 4: S5MT; Lane5: S5GT; Lane 6: H6MT; Lane 7: P6GT; Lane 8: P6MS; Lane 9: P6MTMet; Lane10: P6MT; Lane 11: P7GT; Lane 12: P6MGT; Lane 13: P6MGT-P; Lane 14:P6MT-P; Lane 15: P6GT-P.

FIG. 57 shows an SDS-PAGE analysis (non-reduced) of samples stored at40° C. for 2 months. Lane 1: PEG-hGH Standard; Lane 3: hGH (1 μg); Lane5: P6MT; Lane 6: H6MT; Lane 7: P6GT; Lane 8: P6MS; Lane 9: P6MTMet; Lane10: P6MT; Lane 11: P7GT; Lane 12: P6MGT; Lane 13: P6MGT-P; Lane 14:P6MT-P; Lane 15: P6GT-P.

FIG. 58 shows an SDS-PAGE analysis (reduced) of samples stored at 40° C.for 2 months. Lane 1: PEG-hGH Standard; Lane 3: hGH (1 μg); Lane 5:P6MT; Lane 6: H6MT; Lane 7: P6GT; Lane 8: P6MS; Lane 9: P6MTMet; Lane10: P6MT; Lane 11: P7GT; Lane 12: P6MGT; Lane 13: P6MGT-P; Lane 14:P6MT-P; Lane 15: P6GT-P.

FIG. 59 shows an SDS-PAGE analysis (non-reduced) of reconstitutedsamples stored for 1 week at 4° C. For FIG. 59, panel A, Lane 1: PEG-hGHStandard; Lane 3: P6MT; Lane 4: S4MT; Lane 5: S5MT; Lane 6: S5GT; Lane7: H6MT; Lane 8: P6GT; Lane 9: P6MA; Lane 10: P6MS. For FIG. 59, panelB, Lane 1: PEG-hGH Standard; Lane 3: P6MTMet; Lane 4: P7MT; Lane 5:P7GT; Lane 6: P6MGT; Lane 7: P6MGT-P; Lane 8: P6MT-P; Lane 9: P6GT-P.

FIG. 60 shows an SDS-PAGE analysis (reduced) of reconstituted samplesstored for 1 week at 4° C. For FIG. 60, panel A, Lane 1: PEG-hGHStandard; Lane 3: P6MT; Lane 4: S4MT; Lane 5: S5MT; Lane 6: S5GT; Lane7: H6MT; Lane 8: P6GT; Lane 9: P6MA; Lane 10: P6MS. For FIG. 60, panelB, Lane 1: PEG-hGH Standard; Lane 3: P6MTMet; Lane 4: P7MT; Lane 5:P7GT; Lane 6: P6MGT; Lane 7: P6MGT-P; Lane 8: P6MT-P; Lane 9: P6GT-P.

FIG. 61 shows an SDS-PAGE analysis (non-reduced) of control samples in aformulation study (agitation/UV controls). For FIG. 61, panel A, Lane 1:PEG-hGH Standard; Lane 3: P6MT; Lane 4: S4MT; Lane 5: S5MT; Lane 6:S5GT; Lane 7: H6MT; Lane 8: P6GT; Lane 9: P6MA; Lane 10: P6MS. For FIG.61, panel B, Lane 1: PEG-hGH Standard; Lane 3: P6MTMet; Lane 4: P7MT;Lane 5: P7GT; Lane 6: P6MGT; Lane 7: P6MGT-P; Lane 8: P6MT-P; Lane 9:P6GT-P.

FIG. 62 shows an SDS-PAGE analysis (reduced) of control samples in aformulation study (agitation/UV controls). For FIG. 62, panel A, Lane 1:PEG-hGH Standard; Lane 3: P6MT; Lane 4: S4MT; Lane 5: S5MT; Lane 6:S5GT; Lane 7: H6MT; Lane 8: P6GT; Lane 9: P6MA; Lane 10: P6MS. For FIG.62, panel B, Lane 1: PEG-hGH Standard; Lane 3: P6MTMet; Lane 4: P7MT;Lane 5: P7GT; Lane 6: P6MGT; Lane 7: P6MGT-P; Lane 8: P6MT-P; Lane 9:P6GT-P.

FIG. 63 shows an SDS-PAGE analysis (non-reduced) of samples agitated for4 hours at ambient room temperature. For FIG. 63, panel A, Lane 1:PEG-hGH Standard; Lane 3: P6MT; Lane 4: S4MT; Lane 5: S5MT; Lane 6:S5GT; Lane 7: H6MT; Lane 8: P6GT; Lane 9: P6MA; Lane 10: P6MS. For FIG.63, Panel B, Lane 1: PEG-hGH Standard; Lane 3: P6MTMet; Lane 4: P7MT;Lane 5: P7GT; Lane 6: P6MGT; Lane 7: P6MGT-P; Lane 8: P6MT-P; Lane 9:P6GT-P.

FIG. 64 shows an SDS-PAGE analysis (reduced) of samples agitated for 4hours at ambient room temperature. For FIG. 64, panel A, Lane 1: PEG-hGHStandard; Lane 3: P6MT; Lane 4: S4MT; Lane 5: S5MT; Lane 6: S5GT; Lane7: H6MT; Lane 8: P6GT; Lane 9: P6MA; Lane 10: P6MS. For FIG. 64, panelB, Lane 1: PEG-hGH Standard; Lane 3: P6MTMet; Lane 4: P7MT; Lane 5:P7GT; Lane 6: P6MGT; Lane 7: P6MGT-P; Lane 8: P6MT-P; Lane 9: P6GT-P.

FIG. 65 shows an SDS-PAGE analysis (non-reduced) of samples exposed toUV light for 4 hours at ambient temperature. For FIG. 65, panel A, Lane1: PEG-hGH Standard; Lane 3: P6MT; Lane 4: S4MT; Lane 5: S5MT; Lane 6:S5GT; Lane 7: H6MT; Lane 8: P6GT; Lane 9: P6MA; Lane 10: P6MS. For FIG.65, panel B, Lane 1: PEG-hGH Standard; Lane 3: P6MTMet; Lane 4: P7MT;Lane 5: P7GT; Lane 6: P6MGT; Lane 7: P6MGT-P; Lane 8: P6MT-P; Lane 9:P6GT-P.

FIG. 66 shows an SDS-PAGE analysis (reduced) of samples exposed to UVlight for 4 hours at ambient temperature. For FIG. 66, panel A, Lane 1:PEG-hGH Standard; Lane 3: P6MT; Lane 4: S4MT; Lane 5: S5MT; Lane 6:S5GT; Lane 7: H6MT; Lane 8: P6GT; Lane 9: P6MA; Lane 10: P6MS. For FIG.66, panel B, Lane 1: PEG-hGH Standard; Lane 3: P6MTMet; Lane 4: P7MT;Lane 5: P7GT; Lane 6: P6MGT; Lane 7: P6MGT-P; Lane 8: P6MT-P; Lane 9:P6GT-P.

FIG. 67 shows an SDS-PAGE analysis (non-reduced) of samples that weresubject to freeze/thaw conditions (H7MT-P). Lane 1: PEG-hGH Standard;Lane 2: hGH (1 μg); Lane 3: H7MT-P 8 mg/mL t=0; Lane 4: H7MT-P 8 mg/mLF/T 1; Lane 5: H7MT-P 8 mg/mL F/T 2; Lane 6: H7MT-P 8 mg/mL F/T 3; Lane7: H7MT-P 8 mg/mL F/T 4; Lane 8: H7MT-P 8 mg/mL F/T 5; Lane 10: H7MT-P14 mg/mL t=0; Lane 11: H7MT-P 14 mg/mL F/T 1; Lane 12: H7MT-P 14 mg/mLF/T 2; Lane 13: H7MT-P 14 mg/mL F/T 3; Lane 14: H7MT-P 14 mg/mL F/T 4;Lane 15: H7MT-P 14 mg/mL F/T 5.

FIG. 68 shows an SDS-PAGE analysis (reduced) of samples that weresubject to freeze/thaw conditions (H7MT-P). Lane 1: PEG-hGH Standard;Lane 2: hGH (1 μg); Lane 3: H7MT-P 8 mg/mL t=0; Lane 4: H7MT-P 8 mg/mLF/T 1; Lane 5: H7MT-P 8 mg/mL F/T 2; Lane 6: H7MT-P 8 mg/mL F/T 3; Lane7: H7MT-P 8 mg/mL F/T 4; Lane 8: H7MT-P 8 mg/mL F/T 5; Lane 10: H7MT-P14 mg/mL t=0; Lane 11: H7MT-P 14 mg/mL F/T 1; Lane 12: H7MT-P 14 mg/mLF/T 2; Lane 13: H7MT-P 14 mg/mL F/T 3; Lane 14: H7MT-P 14 mg/mL F/T 4;Lane 15: H7MT-P 14 mg/mL F/T 5.

FIG. 69 shows an SDS-PAGE analysis (non-reduced) of samples that weresubject to freeze/thaw conditions (H7MGT-P). Lane 1: PEG-hGH Standard;Lane 2: hGH (1 μg); Lane 3: H7MGT-P 8 mg/mL t=0; Lane 4: H7MGT-P 8 mg/mLF/T 1; Lane 5: H7MGT-P 8 mg/mL F/T 2; Lane 6: H7MGT-P 8 mg/mL F/T 3;Lane 7: H7MGT-P 8 mg/mL F/T 4; Lane 8: H7MGT-P 8 mg/mL F/T 5; Lane 10:H7MGT-P 14 mg/mL t=0; Lane 11: H7MGT-P 14 mg/mL F/T 1; Lane 12: H7MGT-P14 mg/mL F/T 2; Lane 13: H7MGT-P 14 mg/mL F/T 3; Lane 14: H7MGT-P 14mg/mL F/T 4; Lane 15: H7MGT-P 14 mg/mL F/T 5.

FIG. 70 shows an SDS-PAGE analysis (reduced) of samples that weresubject to freeze/thaw conditions (H7MGT-P). Lane 1: PEG-hGH Standard;Lane 2: hGH (1 μg); Lane 3: H7MGT-P 8 mg/mL t=0; Lane 4: H7MGT-P 8 mg/mLF/T 1; Lane 5: H7MGT-P 8 mg/mL F/T 2; Lane 6: H7MGT-P 8 mg/mL F/T 3;Lane 7: H7MGT-P 8 mg/mL F/T 4; Lane 8: H7MGT-P 8 mg/mL F/T 5; Lane 10:H7MGT-P 14 mg/mL t=0; Lane 11: H7MGT-P 14 mg/mL F/T 1; Lane 12: H7MGT-P14 mg/mL F/T 2; Lane 13: H7MGT-P 14 mg/mL F/T 3; Lane 14: H7MGT-P 14mg/mL F/T 4; Lane 15: H7MGT-P 14 mg/mL F/T 5.

FIG. 71 shows an SDS-PAGE analysis (non-reduced) of control samples andsamples that were subject to agitation for 6 hours and UV light for 4hours. Lane 1: PEG-hGH Standard; Lane 2: hGH (1 μg); Lane 4: Vortex/UVControl H7MT-P 8 mg/mL; Lane 5: Vortex/UV Control H7MT-P 14 mg/mL; Lane6: Vortex/UV Control H7MGT-P 8 mg/mL; Lane 7: Vortex/UV Control H7MGT-P14 mg/mL; Lane 8: Vortex H7MT-P 8 mg/mL; Lane 9: Vortex H7MT-P 14 mg/mL;Lane 10: Vortex H7MGT-P 8 mg/mL; Lane 11: Vortex H7MGT-P 14 mg/mL; Lane12: UV H7MT-P 8 mg/mL; Lane 13: UV H7MT-P 14 mg/mL; Lane 14: UV H7MGT-P8 mg/mL; Lane 15: UV H7MGT-P 14 mg/mL.

FIG. 72 shows an SDS-PAGE analysis (reduced) of control samples andsamples that were subject to agitation for 6 hours and UV light for 4hours. Lane 1: PEG-hGH Standard; Lane 2: hGH (1 μg); Lane 4: Vortex/UVControl H7MT-P 8 mg/mL; Lane 5: Vortex/UV Control H7MT-P 14 mg/mL; Lane6: Vortex/UV Control H7MGT-P 8 mg/mL; Lane 7: Vortex/UV Control H7MGT-P14 mg/mL; Lane 8: Vortex H7MT-P 8 mg/mL; Lane 9: Vortex H7MT-P 14 mg/mL;Lane 10: Vortex H7MGT-P 8 mg/mL* (contaminated); Lane 11: Vortex H7MGT-P14 mg/mL; Lane 12: UV H7MT-P 8 mg/mL; Lane 13: UV H7MT-P 14 mg/mL; Lane14: UV H7MGT-P 8 mg/mL; Lane 15: UV H7MGT-P 14 mg/mL.

FIG. 73 shows an SDS-PAGE analysis (non-reduced) of samples stored at 4°C. or 40° C. for 1 week and samples that were exposed to thermalunfolding conditions. Lane 1: PEG-hGH Standard; Lane 2: hGH (1 μg); Lane4: H7MT-P 8 mg/mL 4° C.; Lane 5: H7MT-P 14 mg/mL 4° C.; Lane 6: H7MGT-P8 mg/mL 4° C.; Lane 7: H7MGT-P 14 mg/mL 4° C.; Lane 8: H7MT-P 8 mg/mL40° C.; Lane 9: H7MT-P 14 mg/mL 40° C.; Lane 10: H7MGT-P 8 mg/mL 40° C.;Lane 11: H7MGT-P 14 mg/mL 40° C.; Lane 12: H7MT-P 8 mg/mLThermal-unfolding; Lane 13: H7MT-P 14 mg/mL Thermal-unfolding; Lane 14:H7MGT-P 8 mg/mL Thermal-unfolding; Lane 15: H7MGT-P 14 mg/mLThermal-unfolding.

FIG. 74 shows an SDS-PAGE analysis (reduced) of samples stored at 4° C.or 40° C. for 1 week and samples that were exposed to thermal unfoldingconditions. Lane 1: PEG-hGH Standard; Lane 2: hGH (1 μg); Lane 4: H7MT-P8 mg/mL 4° C.; Lane 5: H7MT-P 14 mg/mL 4° C.; Lane 6: H7MGT-P 8 mg/mL 4°C.; Lane 7: H7MGT-P 14 mg/mL 4° C.; Lane 8: H7MT-P 8 mg/mL 40° C.; Lane9: H7MT-P 14 mg/mL 40° C.; Lane 10: H7MGT-P 8 mg/mL 40° C.; Lane 11:H7MGT-P 14 mg/mL 40° C.; Lane 12: H7MT-P 8 mg/mL Thermal-unfolding; Lane13: H7MT-P 14 mg/mL Thermal-unfolding; Lane 14: H7MGT-P 8 mg/mLThermal-unfolding; Lane 15: H7MGT-P 14 mg/mL Thermal-unfolding.

FIG. 75 shows an SDS-PAGE analysis (non-reduced) of samples stored at 4°C. or 40° C. for 2 weeks. Lane 1: PEG-hGH Standard; Lane 2: hGH (1 μg);Lane 3: H7MT-P 8 mg/mL 4° C.; Lane 4: H7MT-P 14 mg/mL 4° C.; Lane 5:H7MGT-P 8 mg/mL 4° C.; Lane 6: H7MGT-P 14 mg/mL 4° C.; Lane 7: H7MT-P 8mg/mL 40° C.; Lane 8: H7MT-P 14 mg/mL 40° C.; Lane 9: H7MGT-P 8 mg/mL40° C.; Lane 10: H7MGT-P 14 mg/mL 40° C.

FIG. 76 shows an SDS-PAGE analysis (reduced) of samples stored at 4° C.or 40° C. for 2 weeks. Lane 1: PEG-hGH Standard; Lane 2: hGH (1 μg);Lane 3: H7MT-P 8 mg/mL 4° C.; Lane 4: H7MT-P 14 mg/mL 4° C.; Lane 5:H7MGT-P 8 mg/mL 4° C.; Lane 6: H7MGT-P 14 mg/mL 4° C.; Lane 7: H7MT-P 8mg/mL 40° C.; Lane 8: H7MT-P 14 mg/mL 40° C.; Lane 9: H7MGT-P 8 mg/mL40° C.; Lane 10: H7MGT-P 14 mg/mL 40° C.

FIG. 77 shows an SDS-PAGE analysis (non-reduced) of samples stored at 4°C. for 4 months. Lane 1: PEG-hGH Standard; Lane 2: hGH (1 μg); Lane 4:P6MT; Lane 5: H6MT; Lane 6: P6GT; Lane 7: P6MS; Lane 8: P6MTMet; Lane 9:P6MT; Lane 10: P7GT; Lane 11: P6MGT; Lane 12: P6MGT-P; Lane 13: P6MT-P;Lane 14: P6GT-P.

FIG. 78 shoes an SDS-PAGE analysis (reduced) of samples stored at 4° C.for 4 months. Lane 1: PEG-hGH Standard; Lane 2: hGH (1 μg); Lane 4:P6MT; Lane 5: H6MT; Lane 6: P6GT; Lane 7: P6MS; Lane 8: P6MTMet; Lane 9:P6MT; Lane 10: P7GT; Lane 11: P6MGT; Lane 12: P6MGT-P; Lane 13: P6MT-P;Lane 14: P6GT-P.

FIG. 79 shows an SDS-PAGE analysis (non-reduced) of samples stored at25° C. for 4 months. Lane 1: PEG-hGH Standard; Lane 2: hGH (1 μg); Lane3: P6MT; Lane 4: H6MT; Lane 5: P6GT; Lane 6: P6MS; Lane 7: P6MTMet; Lane8: P6MT; Lane 9: P7GT; Lane 10: P6MGT; Lane 11: P6MGT-P; Lane 12:P6MT-P; Lane 13: P6GT-P.

FIG. 80 shows an SDS-PAGE analysis (reduced) of samples stored at 25° C.for 4 months. Lane 1: PEG-hGH Standard; Lane 2: hGH (1 μg); Lane 3:P6MT; Lane 4: H6MT; Lane 5: P6GT; Lane 6: P6MS; Lane 7: P6MTMet; Lane 8:P6MT; Lane 9: P7GT; Lane 10: P6MGT; Lane 11: P6MGT-P; Lane 12: P6MT-P;Lane 13: P6GT-P.

FIG. 81 shows an SDS-PAGE analysis (non-reduced) of samples stored at 4°C. or 40° C. for 4 weeks. Lane 1: PEG-hGH Standard; Lane 2: hGH (0.5μg); Lane 3: H7MT-P 8 mg/mL 4° C.; Lane 4: H7MT-P 14 mg/mL 4° C.; Lane5: H7MGT-P 8 mg/mL 4° C.; Lane 6: H7MGT-P 14 mg/mL 4° C.; Lane 7: H7MT-P8 mg/mL 40° C.; Lane 8: H7MT-P 14 mg/mL 40° C.; Lane 9: H7MGT-P 8 mg/mL40° C.; Lane 10: H7MGT-P 14 mg/mL 40° C.

FIG. 82 shows an SDS-PAGE analysis (non-reduced) of samples stored at 4°C. or 40° C. for 4 weeks. Lane 1: PEG-hGH Standard; Lane 2: hGH (0.5μg); Lane 3: H7MT-P 8 mg/mL 4° C.; Lane 4: H7MT-P 14 mg/mL 4° C.; Lane5: H7MGT-P 8 mg/mL 4° C.; Lane 6: H7MGT-P 14 mg/mL 4° C.; Lane 7: H7MT-P8 mg/mL 40° C.; Lane 8: H7MT-P 14 mg/mL 40° C.; Lane 9: H7MGT-P 8 mg/mL40° C.; Lane 10: H7MGT-P 14 mg/mL 40° C.

FIG. 83 shows an SDS-PAGE analysis of samples. Lane 1: PEG-hGH Standard(Batch 2); Lane 2: hGH (1 μg); Lane 4: 39.9 mg/mL Non-reduced; Lane 5:24.3 mg/mL Non-reduced; Lane 6: 1.1 mg/mL Non-reduced; Lane 8: 39.9mg/mL Reduced; Lane 9: 24.3 mg/mL Reduced; Lane 10:1.1 mg/mL Reduced.

DEFINITIONS

It is to be understood that this invention is not limited to theparticular methodology, protocols, cell lines, constructs, and reagentsdescribed herein and as such may vary. It is also to be understood thatthe terminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention, which will be limited only by the appended claims.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural reference unless the context clearly indicatesotherwise. Thus, for example, reference to a “hGH” is a reference to oneor more such proteins and includes equivalents thereof known to those ofordinary skill in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs. Although any methods, devices,and materials similar or equivalent to those described herein can beused in the practice or testing of the invention, the preferred methods,devices and materials are now described.

All publications and patents mentioned herein are incorporated herein byreference for the purpose of describing and disclosing, for example, theconstructs and methodologies that are described in the publications,which might be used in connection with the presently describedinvention. The publications discussed herein are provided solely fortheir disclosure prior to the filing date of the present application.Nothing herein is to be construed as an admission that the inventors arenot entitled to antedate such disclosure by virtue of prior invention orfor any other reason.

U.S. patent application Ser. No. 11/046,432 is incorporated by referencein its entirety. Thus, the disclosures provided in paragraphs numbered79-153, in U.S. patent application Ser. No. 11/046,432 apply fully tothe methods, compositions, techniques and strategies for making,purifying, characterizing, and using non-natural amino acids,non-natural amino acid hGH polypeptides and modified non-natural aminoacid hGH polypeptides described herein to the same extent as if suchdisclosures were fully presented herein.

As used herein, “growth hormone” or “GH” shall include thosepolypeptides and proteins that have at least one biological activity ofa human growth hormone, as well as GH analogs, GH isoforms, GH mimetics,GH fragments, hybrid GH proteins, fusion proteins, oligomers andmultimers, homologues, glycosylation pattern variants, variants, splicevariants, and muteins, thereof, regardless of the biological activity ofsame, and further regardless of the method of synthesis or manufacturethereof including, but not limited to, recombinant (whether producedfrom cDNA, genomic DNA, synthetic DNA or other form of nucleic acid), invitro, in vivo, by microinjection of nucleic acid molecules, synthetic,transgenic, and gene activated methods. The term “hGH polypeptide”encompasses hGH polypeptides comprising one or more amino acidsubstitutions, additions or deletions.

For the complete full-length naturally-occurring GH amino acid sequenceas well as the mature naturally-occurring GH amino acid sequence andnaturally occurring mutant, see SEQ ID NO: 1, SEQ ID NO: 2 and SEQ IDNO: 3, respectively, in U.S. patent application Ser. No. 11/046,432,which is incorporated by reference herein. In some embodiments, hGHpolypeptides of the invention are substantially identical to thesesequences or any other sequence of a growth hormone polypeptide.

The term “hGH polypeptide” also includes the pharmaceutically acceptablesalts and prodrugs, and prodrugs of the salts, polymorphs, hydrates,solvates, biologically-active fragments, biologically active variantsand stereoisomers of the naturally-occurring hGH as well as agonist,mimetic, and antagonist variants of the naturally-occurring hGH andpolypeptide fusions thereof. Fusions comprising additional amino acidsat the amino terminus, carboxyl terminus, or both, are encompassed bythe term “hGH polypeptide.” Exemplary fusions include, but are notlimited to, e.g., methionyl growth hormone in which a methionine islinked to the N-terminus of hGH resulting from the recombinantexpression, fusions for the purpose of purification (including, but notlimited to, to poly-histidine or affinity epitopes), fusions with serumalbumin binding peptides and fusions with serum proteins such as serumalbumin. U.S. Pat. No. 5,750,373, which is incorporated by referenceherein, describes a method for selecting novel proteins such as growthhormone and antibody fragment variants having altered binding propertiesfor their respective receptor molecules. The method comprises fusing agene encoding a protein of interest to the carboxy terminal domain ofthe gene III coat protein of the filamentous phage M13.

Various references disclose modification of polypeptides by polymerconjugation or glycosylation. The term “hGH polypeptide” includespolypeptides conjugated to a polymer such as PEG and may be comprised ofone or more additional derivitizations of cysteine, lysine, or otherresidues. In addition, the hGH polypeptide may comprise a linker orpolymer, wherein the amino acid to which the linker or polymer isconjugated may be a non-natural amino acid according to the presentinvention, or may be conjugated to a naturally encoded amino acidutilizing techniques known in the art such as coupling to lysine orcysteine.

Polymer conjugation of hGH polypeptides has been reported. See, e.g.U.S. Pat. Nos. 5,849,535, 6,136,563 and 6,608,183, which areincorporated by reference herein. U.S. Pat. No. 4,904,584 disclosesPEGylated lysine depleted polypeptides, wherein at least one lysineresidue has been deleted or replaced with any other amino acid residue.WO 99/67291 discloses a process for conjugating a protein with PEG,wherein at least one amino acid residue on the protein is deleted andthe protein is contacted with PEG under conditions sufficient to achieveconjugation to the protein. WO 99/03887 discloses PEGylated variants ofpolypeptides belonging to the growth hormone superfamily, wherein acysteine residue has been substituted with a non-essential amino acidresidue located in a specified region of the polypeptide. WO 00/26354discloses a method of producing a glycosylated polypeptide variant withreduced allergenicity, which as compared to a corresponding parentpolypeptide comprises at least one additional glycosylation site. U.S.Pat. No. 5,218,092, which is incorporated by reference herein, disclosesmodification of granulocyte colony stimulating factor (G-CSF) and otherpolypeptides so as to introduce at least one additional carbohydratechain as compared to the native polypeptide.

The term “hGH polypeptide” also includes glycosylated hGH, such as butnot limited to, polypeptides glycosylated at any amino acid position,N-linked or O-linked glycosylated forms of the polypeptide. Variantscontaining single nucleotide changes are also considered as biologicallyactive variants of hGH polypeptide. In addition, splice variants arealso included. The term “hGH polypeptide” also includes hGH polypeptideheterodimers, homodimers, heteromultimers, or homomultimers of any oneor more hGH polypeptides or any other polypeptide, protein,carbohydrate, polymer, small molecule, linker, ligand, or otherbiologically active molecule of any type, linked by chemical means orexpressed as a fusion protein, as well as polypeptide analoguescontaining, for example, specific deletions or other modifications yetmaintain biological activity.

All references to amino acid positions in hGH described herein are basedon the position in SEQ ID NO: 2 as listed in U.S. patent applicationSer. No. 11/046,432, entitled “Modified Human Growth HormonePolypeptides and Their Uses,” which is incorporated by reference herein,unless otherwise specified (i.e., when it is stated that the comparisonis based on another hGH sequence such as SEQ ID NO: 1, 3). Those ofskill in the art will appreciate that amino acid positions correspondingto positions in SEQ ID NO: 1, 2, 3 listed in U.S. patent applicationSer. No. 11/046,432, which is incorporated by reference herein, or anyother GH sequence can be readily identified in any other hGH moleculesuch as hGH fusions, variants, fragments, etc. For example, sequencealignment programs such as BLAST can be used to align and identify aparticular position in a protein that corresponds with a position in SEQID NO: 1, 2, 3 of U.S. patent application Ser. No. 11/046,432, which isincorporated by reference herein, or other GH sequence. Substitutions,deletions or additions of amino acids described herein in reference toSEQ ID NO: 1, 2, 3 of U.S. patent application Ser. No. 11/046,432, whichis incorporated by reference herein, or other GH sequence are intendedto also refer to substitutions, deletions or additions in correspondingpositions in any other hGH molecule such as hGH fusions, variants,fragments, etc. described herein or known in the art and are expresslyencompassed by the present invention.

The term “hGH polypeptide” or “hGH” encompasses hGH polypeptidescomprising one or more amino acid substitutions, additions or deletions.hGH polypeptides of the present invention may be comprised ofmodifications with one or more natural amino acids in conjunction withone or more non-natural amino acid modification. Exemplary substitutionsin a wide variety of amino acid positions in naturally-occurring hGHpolypeptides have been described, including but not limited tosubstitutions that modulate one or more of the biological activities ofthe hGH polypeptide, such as but not limited to, increase agonistactivity, increase solubility of the polypeptide, decrease proteasesusceptibility, convert the polypeptide into an antagonist, etc. and areencompassed by the term “hGH polypeptide.”

In some embodiments, the hGH polypeptides further comprise an addition,substitution or deletion that modulates biological activity of the hGHpolypeptide. For example, the additions, substitutions or deletions maymodulate one or more properties or activities of hGH. For example, theadditions, substitutions or deletions may modulate affinity for the hGHpolypeptide receptor, modulate (including but not limited to, increasesor decreases) receptor dimerization, stabilize receptor dimers, modulatecirculating half-life, modulate therapeutic half-life, modulatestability of the polypeptide, modulate cleavage by proteases, modulatedose, modulate release or bio-availability, facilitate purification, orimprove or alter a particular route of administration. Similarly, hGHpolypeptides may comprise protease cleavage sequences, secretion signalsequences, reactive groups, antibody-binding domains (including but notlimited to, FLAG or poly-His) or other affinity based sequences(including but not limited to, FLAG, poly-His, GST, etc.) or linkedmolecules (including but not limited to, biotin) that improve detection(including but not limited to, GFP), purification or other traits of thepolypeptide.

The term “hGH polypeptide” also encompasses homodimers, heterodimers,homomultimers, and heteromultimers that are linked, including but notlimited to those linked directly via non-naturally encoded amino acidside chains, either to the same or different non-naturally encoded aminoacid side chains, to naturally-encoded amino acid side chains, orindirectly via a linker. Exemplary linkers including but are not limitedto, small organic compounds, water soluble polymers of a variety oflengths such as poly(ethylene glycol) or polydextran, or polypeptides ofvarious lengths.

A “non-naturally encoded amino acid” refers to an amino acid that is notone of the 20 common amino acids or pyrrolysine or selenocysteine. Otherterms that may be used synonymously with the term “non-naturally encodedamino acid” are “non-natural amino acid,” “unnatural amino acid,”“non-naturally-occurring amino acid,” and variously hyphenated andnon-hyphenated versions thereof. The term “non-naturally encoded aminoacid” also includes, but is not limited to, amino acids that occur bymodification (e.g. post-translational modifications) of a naturallyencoded amino acid (including but not limited to, the 20 common aminoacids or pyrrolysine and selenocysteine) but are not themselvesnaturally incorporated into a growing polypeptide chain by thetranslation complex. Examples of such non-naturally-occurring aminoacids include, but are not limited to, N-acetylglucosaminyl-L-serine,N-acetylglucosaminyl-L-threonine, and O-phosphotyrosine.

An “amino terminus modification group” refers to any molecule that canbe attached to the amino terminus of a polypeptide. Similarly, a“carboxy terminus modification group” refers to any molecule that can beattached to the carboxy terminus of a polypeptide. Terminus modificationgroups include, but are not limited to, various water soluble polymers,peptides or proteins such as serum albumin, or other moieties thatincrease serum half-life of peptides.

The terms “functional group”, “active moiety”, “activating group”,“leaving group”, “reactive site”, “chemically reactive group” and“chemically reactive moiety” are used in the art and herein to refer todistinct, definable portions or units of a molecule. The terms aresomewhat synonymous in the chemical arts and are used herein to indicatethe portions of molecules that perform some function or activity and arereactive with other molecules.

The term “linkage” or “linker” is used herein to refer to groups orbonds that normally are formed as the result of a chemical reaction andtypically are covalent linkages. Hydrolytically stable linkages meansthat the linkages are substantially stable in water and do not reactwith water at useful pH values, including but not limited to, underphysiological conditions for an extended period of time, perhaps evenindefinitely. Hydrolytically unstable or degradable linkages mean thatthe linkages are degradable in water or in aqueous solutions, includingfor example, blood. Enzymatically unstable or degradable linkages meanthat the linkage can be degraded by one or more enzymes. As understoodin the art, PEG and related polymers may include degradable linkages inthe polymer backbone or in the linker group between the polymer backboneand one or more of the terminal functional groups of the polymermolecule. For example, ester linkages formed by the reaction of PEGcarboxylic acids or activated PEG carboxylic acids with alcohol groupson a biologically active agent generally hydrolyze under physiologicalconditions to release the agent. Other hydrolytically degradablelinkages include, but are not limited to, carbonate linkages; iminelinkages resulted from reaction of an amine and an aldehyde; phosphateester linkages formed by reacting an alcohol with a phosphate group;hydrazone linkages which are reaction product of a hydrazide and analdehyde; acetal linkages that are the reaction product of an aldehydeand an alcohol; orthoester linkages that are the reaction product of aformate and an alcohol; peptide linkages formed by an amine group,including but not limited to, at an end of a polymer such as PEG, and acarboxyl group of a peptide; and oligonucleotide linkages formed by aphosphoramidite group, including but not limited to, at the end of apolymer, and a 5′ hydroxyl group of an oligonucleotide.

The term “biologically active molecule”, “biologically active moiety” or“biologically active agent” when used herein means any substance whichcan affect any physical or biochemical properties of a biologicalsystem, pathway, molecule, or interaction relating to an organism,including but not limited to, viruses, bacteria, bacteriophage,transposon, prion, insects, fungi, plants, animals, and humans. Inparticular, as used herein, biologically active molecules include, butare not limited to, any substance intended for diagnosis, cure,mitigation, treatment, or prevention of disease in humans or otheranimals, or to otherwise enhance physical or mental well-being of humansor animals. Examples of biologically active molecules include, but arenot limited to, peptides, proteins, enzymes, small molecule drugs, harddrugs, soft drugs, carbohydrates, inorganic atoms or molecules, dyes,lipids, nucleosides, radionuclides, oligonucleotides, toxins, cells,viruses, liposomes, microparticles and micelles. Classes of biologicallyactive agents that are suitable for use with the invention include, butare not limited to, drugs, prodrugs, radionuclides, imaging agents,polymers, antibiotics, fungicides, anti-viral agents, anti-inflammatoryagents, anti-tumor agents, cardiovascular agents, anti-anxiety agents,hormones, growth factors, steroidal agents, microbially derived toxins,and the like.

A “bifunctional polymer” refers to a polymer comprising two discretefunctional groups that are capable of reacting specifically with othermoieties (including but not limited to, amino acid side groups) to formcovalent or non-covalent linkages. A bifunctional linker having onefunctional group reactive with a group on a particular biologicallyactive component, and another group reactive with a group on a secondbiological component, may be used to form a conjugate that includes thefirst biologically active component, the bifunctional linker and thesecond biologically active component. Many procedures and linkermolecules for attachment of various compounds to peptides are known.See, e.g., European Patent Application No. 188,256; U.S. Pat. Nos.4,671,958, 4,659,839, 4,414,148, 4,699,784; 4,680,338; and 4,569,789which are incorporated by reference herein. A “multi-functional polymer”refers to a polymer comprising two or more discrete functional groupsthat are capable of reacting specifically with other moieties (includingbut not limited to, amino acid side groups) to form covalent ornon-covalent linkages. A bi-functional polymer or multifunctionalpolymer may be any desired length or molecular weight, and may beselected to provide a particular desired spacing or conformation betweenone or more molecules linked to the GH, e.g., hGH, molecule.

As used herein, the term “water soluble polymer” refers to any polymerthat is soluble in aqueous solvents. Linkage of water soluble polymersto hGH polypeptides can result in changes including, but not limited to,increased or modulated serum half-life, or increased or modulatedtherapeutic half-life relative to the unmodified form, modulatedimmunogenicity, modulated physical association characteristics such asaggregation and multimer formation, altered receptor binding and alteredreceptor dimerization or multimerization. The water soluble polymer mayor may not have its own biological activity. Suitable polymers include,but are not limited to, polyethylene glycol, polyethylene glycolpropionaldehyde, mono C1-C10 alkoxy or aryloxy derivatives thereof(described in U.S. Pat. No. 5,252,714 which is incorporated by referenceherein), monomethoxy-polyethylene glycol, polyvinyl pyrrolidone,polyvinyl alcohol, polyamino acids, divinylether maleic anhydride,N-(2-Hydroxypropyl)-methacrylamide, dextran, dextran derivativesincluding dextran sulfate, polypropylene glycol, polypropyleneoxide/ethylene oxide copolymer, polyoxyethylated polyol, heparin,heparin fragments, polysaccharides, oligosaccharides, glycans, celluloseand cellulose derivatives, including but not limited to methylcelluloseand carboxymethyl cellulose, starch and starch derivatives,polypeptides, polyalkylene glycol and derivatives thereof, copolymers ofpolyalkylene glycols and derivatives thereof, polyvinyl ethyl ethers,and alpha-beta-poly[(2-hydroxyethyl)-DL-aspartamide, and the like, ormixtures thereof. Examples of such water soluble polymers include, butare not limited to, polyethylene glycol and serum albumin.

As used herein, the term “polyalkylene glycol” or “poly(alkene glycol)”refers to polyethylene glycol (poly(ethylene glycol)), polypropyleneglycol, polybutylene glycol, and derivatives thereof. The term“polyalkylene glycol” encompasses both linear and branched polymers andaverage molecular weights of between 0.1 kDa and 100 kDa. Otherexemplary embodiments are listed, for example, in commercial suppliercatalogs, such as Shearwater Corporation's catalog “Polyethylene Glycoland Derivatives for Biomedical Applications” (2001).

As used herein, the term “modulated serum half-life” means the positiveor negative change in circulating half-life of a modified hGH relativeto its non-modified form. Serum half-life is measured by taking bloodsamples at various time points after administration of hGH, anddetermining the concentration of that molecule in each sample.Correlation of the serum concentration with time allows calculation ofthe serum half-life. Increased serum half-life desirably has at leastabout two-fold, but a smaller increase may be useful, for example whereit enables a satisfactory dosing regimen or avoids a toxic effect. Insome embodiments, the increase is at least about three-fold, at leastabout five-fold, or at least about ten-fold.

The term “modulated therapeutic half-life” as used herein means thepositive or negative change in the half-life of the therapeuticallyeffective amount of hGH, relative to its non-modified form. Therapeutichalf-life is measured by measuring pharmacokinetic and/orpharmacodynamic properties of the molecule at various time points afteradministration. Increased therapeutic half-life desirably enables aparticular beneficial dosing regimen, a particular beneficial totaldose, or avoids an undesired effect. In some embodiments, the increasedtherapeutic half-life results from increased potency, increased ordecreased binding of the modified molecule to its target, increased ordecreased breakdown of the molecule by enzymes such as proteases, or anincrease or decrease in another parameter or mechanism of action of thenon-modified molecule.

The term “substantially purified” refers to a hGH polypeptide that maybe substantially or essentially free of components that normallyaccompany or interact with the protein as found in its naturallyoccurring environment, i.e. a native cell, or host cell in the case ofrecombinantly produced hGH polypeptides. hGH polypeptide that may besubstantially free of cellular material includes preparations of proteinhaving less than about 30%, less than about 25%, less than about 20%,less than about 15%, less than about 10%, less than about 5%, less thanabout 4%, less than about 3%, less than about 2%, or less than about 1%(by dry weight) of contaminating protein. When the hGH polypeptide orvariant thereof is recombinantly produced by the host cells, the proteinmay be present at about 30%, about 25%, about 20%, about 15%, about 10%,about 5%, about 4%, about 3%, about 2%, or about 1% or less of the dryweight of the cells. When the hGH polypeptide or variant thereof isrecombinantly produced by the host cells, the protein may be present inthe culture medium at about 5 g/L, about 4 g/L, about 3 g/L, about 2g/L, about 1 g/L, about 750 mg/L, about 500 mg/L, about 250 mg/L, about100 mg/L, about 50 mg/L, about 10 mg/L, or about 1 mg/L or less of thedry weight of the cells. Thus, “substantially purified” hGH polypeptideas produced by the methods of the present invention may have a puritylevel of at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, at least about 55%, at least about60%, at least about 65%, at least about 70%, specifically, a puritylevel of at least about 75%, 80%, 85%, and more specifically, a puritylevel of at least about 90%, a purity level of at least about 95%, apurity level of at least about 99% or greater as determined byappropriate methods such as SDS/PAGE analysis, RP-HPLC, SEC, andcapillary electrophoresis.

The term “isolated,” when applied to a nucleic acid or protein, denotesthat the nucleic acid or protein is free of at least some of thecellular components with which it is associated in the natural state, orthat the nucleic acid or protein has been concentrated to a levelgreater than the concentration of its in vivo or in vitro production. Itcan be in a homogeneous state. Isolated substances can be in either adry or semi-dry state, or in solution, including but not limited to, anaqueous solution. It can be a component of a pharmaceutical compositionthat comprises additional pharmaceutically acceptable carriers and/orexcipients. Purity and homogeneity are typically determined usinganalytical chemistry techniques such as polyacrylamide gelelectrophoresis or high performance liquid chromatography. A proteinwhich is the predominant species present in a preparation issubstantially purified. In particular, an isolated gene is separatedfrom open reading frames which flank the gene and encode a protein otherthan the gene of interest. The term “purified” denotes that a nucleicacid or protein gives rise to substantially one band in anelectrophoretic gel. Particularly, it may mean that the nucleic acid orprotein is at least 85% pure, at least 90% pure, at least 95% pure, atleast 99% or greater pure.

The term “subject” as used herein, refers to an animal, in someembodiments a mammal, and in other embodiments a human, who is theobject of treatment, observation or experiment.

The term “effective amount” as used herein refers to that amount of themodified non-natural amino acid polypeptide being administered whichwill relieve to some extent one or more of the symptoms of the disease,condition or disorder being treated. Compositions containing themodified non-natural amino acid polypeptide described herein can beadministered for prophylactic, enhancing, and/or therapeutic treatments.

The terms “enhance” or “enhancing” means to increase or prolong eitherin potency or duration a desired effect. Thus, in regard to enhancingthe effect of therapeutic agents, the term “enhancing” refers to theability to increase or prolong, either in potency or duration, theeffect of other therapeutic agents on a system. An “enhancing-effectiveamount,” as used herein, refers to an amount adequate to enhance theeffect of another therapeutic agent in a desired system. When used in apatient, amounts effective for this use will depend on the severity andcourse of the disease, disorder or condition, previous therapy, thepatient's health status and response to the drugs, and the judgment ofthe treating physician.

The term “modified,” as used herein refers to any changes made to agiven polypeptide, such as changes to the length of the polypeptide, theamino acid sequence, chemical structure, co-translational modification,or post-translational modification of a polypeptide. The form“(modified)” term means that the polypeptides being discussed areoptionally modified, that is, the polypeptides under discussion can bemodified or unmodified.

The term “post-translationally modified” refers to any modification of anatural or non-natural amino acid that occurs to such an amino acidafter it has been incorporated into a polypeptide chain. The termencompasses, by way of example only, co-translational in vivomodifications, co-translational in vitro modifications (such as in acell-free translation system), post-translational in vivo modifications,and post-translational in vitro modifications.

In prophylactic applications, compositions containing the modifiednon-natural amino acid polypeptide are administered to a patientsusceptible to or otherwise at risk of a particular disease, disorder orcondition. Such an amount is defined to be a “prophylactically effectiveamount.” In this use, the precise amounts also depend on the patient'sstate of health, weight, and the like. It is considered well within theskill of the art for one to determine such prophylactically effectiveamounts by routine experimentation (e.g., a dose escalation clinicaltrial).

In therapeutic applications, compositions containing the modifiednon-natural amino acid polypeptide are administered to a patient alreadysuffering from a disease, condition or disorder, in an amount sufficientto cure or at least partially arrest the symptoms of the disease,disorder or condition. Such an amount is defined to be a“therapeutically effective amount,” and will depend on the severity andcourse of the disease, disorder or condition, previous therapy, thepatient's health status and response to the drugs, and the judgment ofthe treating physician. It is considered well within the skill of theart for one to determine such therapeutically effective amounts byroutine experimentation (e.g., a dose escalation clinical trial).

The term “treating” is used to refer to either prophylactic and/ortherapeutic treatments.

Non-naturally encoded amino acid polypeptides may be metabolized uponadministration to an organism in need to produce a metabolite that isthen used to produce a desired effect, including a desired therapeuticeffect.

Unless otherwise indicated, conventional methods of mass spectroscopy,NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniquesand pharmacology, within the skill of the art are employed.

DETAILED DESCRIPTION I. Introduction

hGH molecules comprising at least one unnatural amino acid are providedin the invention. In certain embodiments of the invention, the hGHpolypeptide with at least one unnatural amino acid includes at least onepost-translational modification. In one embodiment, the at least onepost-translational modification comprises attachment of a moleculeincluding but not limited to, a label, a dye, a polymer, a water-solublepolymer, a derivative of polyethylene glycol, a photocrosslinker, aradionuclide, a cytotoxic compound, a drug, an affinity label, aphotoaffinity label, a reactive compound, a resin, a second protein orpolypeptide or polypeptide analog, an antibody or antibody fragment, ametal chelator, a cofactor, a fatty acid, a carbohydrate, apolynucleotide, a DNA, a RNA, an antisense polynucleotide, a saccharide,a water-soluble dendrimer, a cyclodextrin, an inhibitory ribonucleicacid, a biomaterial, a nanoparticle, a spin label, a fluorophore, ametal-containing moiety, a radioactive moiety, a novel functional group,a group that covalently or noncovalently interacts with other molecules,a photocaged moiety, an actinic radiation excitable moiety, aphotoisomerizable moiety, biotin, a derivative of biotin, a biotinanalogue, a moiety incorporating a heavy atom, a chemically cleavablegroup, a photocleavable group, an elongated side chain, a carbon-linkedsugar, a redox-active agent, an amino thioacid, a toxic moiety, anisotopically labeled moiety, a biophysical probe, a phosphorescentgroup, a chemiluminescent group, an electron dense group, a magneticgroup, an intercalating group, a chromophore, an energy transfer agent,a biologically active agent, a detectable label, a small molecule, aquantum dot, a nanotransmitter, a radionucleotide, a radiotransmitter, aneutron-capture agent, or any combination of the above or any otherdesirable compound or substance, comprising a second reactive group toat least one unnatural amino acid comprising a first reactive grouputilizing chemistry methodology that is known to one of ordinary skillin the art to be suitable for the particular reactive groups.

The protein or polypeptide of interest can contain at least one, atleast two, at least three, at least four, at least five, at least six,at least seven, at least eight, at least nine, or ten or more unnaturalamino acids. The unnatural amino acids can be the same or different, forexample, there can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more differentsites in the protein that comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or moredifferent unnatural amino acids. In certain embodiments, at least one,but fewer than all, of a particular amino acid present in a naturallyoccurring version of the protein are substituted with an unnatural aminoacid.

The present invention provides methods and compositions based on membersof the GH supergene family, in particular hGH, comprising at least onenon-naturally encoded amino acid. Introduction of at least onenon-naturally encoded amino acid into a GH supergene family member suchas hGH can allow for the application of conjugation chemistries thatinvolve specific chemical reactions, including, but not limited to, withone or more non-naturally encoded amino acids while not reacting withthe commonly occurring 20 amino acids. In some embodiments, the GHsupergene family member comprising the non-naturally encoded amino acidis linked to a water soluble polymer, such as polyethylene glycol (PEG),via the side chain of the non-naturally encoded amino acid. Thisinvention provides a highly efficient method for the selectivemodification of proteins with PEG derivatives, which involves theselective incorporation of non-genetically encoded amino acids,including but not limited to, those amino acids containing functionalgroups or substituents not found in the 20 naturally incorporated aminoacids, including but not limited to a ketone, an azide or acetylenemoiety, into proteins in response to a selector codon and the subsequentmodification of those amino acids with a suitably reactive PEGderivative. Once incorporated, the amino acid side chains can then bemodified by utilizing chemistry methodologies known to those of ordinaryskill in the art to be suitable for the particular functional groups orsubstituents present in the non-naturally encoded amino acid. Knownchemistry methodologies of a wide variety are suitable for use in thepresent invention to incorporate a water soluble polymer into theprotein.

It is well established in the art that PEG can be used to modify thesurfaces of biomaterials (see, e.g., U.S. Pat. No. 6,610,281; Mehvar,R., J. Pharmaceut. Sci., 3(1):125-136 (2000) which are incorporated byreference herein).

A discussion of recombinant nucleic acid methods, selector codons,orthogonal tRNAs, orthogonal aminoacyl tRNA synthetases, andnon-naturally encoded amino acids with various reactive groups,including but not limited to, carbonyl groups, hydrazine, hydrazide,aminooxy, azide, and alkyne groups, is provided in U.S. patentapplication Ser. No. 11/046,432 entitled “Modified Human Growth HormonePolypeptides and Their Uses,” which is incorporated by reference in itsentirety herein. Cellular uptake and biosynthesis of non-naturallyencoded amino acids are also discussed in this application. Thisapplication also details sites for incorporation of one or morenon-naturally encoded amino acids into hGH and expression of hGHpolypeptides. The synthesis of non-natural amino acids containingcarbonyl groups such as p-acetyl-(+/−)-phenylalanine andm-acetyl-(+/−)-phenylalanine is described in Zhang, Z., et al.,Biochemistry 42: 6735-6746 (2003), which is incorporated by referenceherein.

II. Polypeptides with Unnatural Amino Acids

The incorporation of an unnatural amino acid can be done for a varietyof purposes, including but not limited to, tailoring changes in proteinstructure and/or function, changing size, acidity, nucleophilicity,hydrogen bonding, hydrophobicity, accessibility of protease targetsites, targeting to a moiety (including but not limited to, for aprotein array), adding a biologically active molecule, attaching apolymer, attaching a radionuclide, modulating serum half-life,modulating tissue penetration (e.g., tumors), modulating activetransport, modulating tissue, cell or organ specificity or distribution,modulating immunogenicity, modulating protease resistance, etc. Proteinsthat include an unnatural amino acid can have enhanced or even entirelynew catalytic or biophysical properties. For example, the followingproperties are optionally modified by inclusion of an unnatural aminoacid into a protein: toxicity, biodistribution, structural properties,spectroscopic properties, chemical and/or photochemical properties,catalytic ability, half-life (including but not limited to, serumhalf-life), ability to react with other molecules, including but notlimited to, covalently or noncovalently, and the like. The compositionsincluding proteins that include at least one unnatural amino acid areuseful for, including but not limited to, novel therapeutics,diagnostics, catalytic enzymes, industrial enzymes, binding proteins(including but not limited to, antibodies), and including but notlimited to, the study of protein structure and function. See, e.g.,Dougherty, (2000) Unnatural Amino Acids as Probes of Protein Structureand Function, Current Opinion in Chemical Biology, 4:645-652.

In one aspect of the invention, a composition includes at least oneprotein with at least one, including but not limited to, at least two,at least three, at least four, at least five, at least six, at leastseven, at least eight, at least nine, or at least ten or more unnaturalamino acids. The unnatural amino acids can be the same or different,including but not limited to, there can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or10 or more different sites in the protein that comprise 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 or more different unnatural amino acids. In anotheraspect, a composition includes a protein with at least one, but fewerthan all, of a particular amino acid present in the protein issubstituted with the unnatural amino acid. For a given protein with morethan one unnatural amino acids, the unnatural amino acids can beidentical or different (including but not limited to, the protein caninclude two or more different types of unnatural amino acids, or caninclude two of the same unnatural amino acid). For a given protein withmore than two unnatural amino acids, the unnatural amino acids can bethe same, different or a combination of a multiple unnatural amino acidof the same kind with at least one different unnatural amino acid.

Proteins or polypeptides of interest with at least one unnatural aminoacid are a feature of the invention. The invention also includespolypeptides or proteins with at least one unnatural amino acid producedusing the compositions and methods of the invention. An excipient(including but not limited to, a pharmaceutically acceptable excipient)can also be present with the protein.

By producing proteins or polypeptides of interest with at least oneunnatural amino acid in eukaryotic cells, proteins or polypeptides willtypically include eukaryotic post-translational modifications. Incertain embodiments, a protein includes at least one unnatural aminoacid and at least one post-translational modification that is made invivo by a eukaryotic cell, where the post-translational modification isnot made by a prokaryotic cell. For example, the post-translationmodification includes, including but not limited to, acetylation,acylation, lipid-modification, palmitoylation, palmitate addition,phosphorylation, glycolipid-linkage modification, glycosylation, and thelike. In one aspect, the post-translational modification includesattachment of an oligosaccharide (including but not limited to,(GlcNAc-Man)₂-Man-GlcNAc-GlcNAc)) to an asparagine by aGlcNAc-asparagine linkage. See Table 1 of U.S. patent application Ser.No. 11/046,432 entitled “Modified Human Growth Hormone Polypeptides andTheir Uses,” which is incorporated by reference herein, which lists someexamples of N-linked oligosaccharides of eukaryotic proteins (additionalresidues can also be present, which are not shown). In another aspect,the post-translational modification includes attachment of anoligosaccharide (including but not limited to, Gal-GalNAc, Gal-GlcNAc,etc.) to a serine or threonine by a GalNAc-serine or GalNAc-threoninelinkage, or a GlcNAc-serine or a GlcNAc-threonine linkage.

In yet another aspect, the post-translation modification includesproteolytic processing of precursors (including but not limited to,calcitonin precursor, calcitonin gene-related peptide precursor,preproparathyroid hormone, preproinsulin, proinsulin,prepro-opiomelanocortin, pro-opiomelanocortin and the like), assemblyinto a multisubunit protein or macromolecular assembly, translation toanother site in the cell (including but not limited to, to organelles,such as the endoplasmic reticulum, the Golgi apparatus, the nucleus,lysosomes, peroxisomes, mitochondria, chloroplasts, vacuoles, etc., orthrough the secretory pathway). In certain embodiments, the proteincomprises a secretion or localization sequence, an epitope tag, a FLAGtag, a polyhistidine tag, a GST fusion, or the like. U.S. Pat. Nos.4,963,495 and 6,436,674, which are incorporated herein by reference,detail constructs designed to improve secretion of hGH polypeptides.

One advantage of an unnatural amino acid is that it presents additionalchemical moieties that can be used to add additional molecules. Thesemodifications can be made in vivo in a eukaryotic or non-eukaryoticcell, or in vitro. Thus, in certain embodiments, the post-translationalmodification is through the unnatural amino acid. For example, thepost-translational modification can be through anucleophilic-electrophilic reaction. Most reactions currently used forthe selective modification of proteins involve covalent bond formationbetween nucleophilic and electrophilic reaction partners, including butnot limited to the reaction of α-haloketones with histidine or cysteineside chains. Selectivity in these cases is determined by the number andaccessibility of the nucleophilic residues in the protein. In proteinsof the invention, other more selective reactions can be used such as thereaction of an unnatural keto-amino acid with hydrazides or aminooxycompounds, in vitro and in vivo. See, e.g., Cornish, et al., (1996) J.Am. Chem. Soc., 118:8150-8151; Mahal, et al., (1997) Science,276:1125-1128; Wang, et al., (2001) Science 292:498-500; Chin, et al.,(2002) J. Am. Chem. Soc. 124:9026-9027; Chin, et al., (2002) Proc. Natl.Acad. Sci., 99:11020-11024; Wang, et al., (2003) Proc. Natl. Acad. Sci.,100:56-61; Zhang, et al., (2003) Biochemistry, 42:6735-6746; and, Chin,et al., (2003) Science, 301:964-7, all of which are incorporated byreference herein. This allows the selective labeling of virtually anyprotein with a host of reagents including fluorophores, crosslinkingagents, saccharide derivatives and cytotoxic molecules. See also, U.S.Pat. No. 6,927,042 entitled “Glycoprotein synthesis,” which isincorporated by reference herein. Molecules that may be attached to hGHinclude, but are not limited to, dyes, fluorophores, crosslinkingagents, saccharide derivatives, polymers (including but not limited to,derivatives of polyethylene glycol), photocrosslinkers, cytotoxiccompounds, affinity labels, derivatives of biotin, resins, beads, asecond protein or polypeptide (or more), polynucleotide(s) (includingbut not limited to, DNA, RNA, etc.), metal chelators, cofactors, fattyacids, carbohydrates, and the like.

This invention provides formulations of non-natural amino acidpolypeptides generated via selective modification of proteins, whichinvolves the genetic incorporation of unnatural amino acids.

III. In Vivo Generation of hGH Polypeptides ComprisingNon-Genetically-Encoded Amino Acids

The hGH polypeptides of the invention can be generated in vivo usingmodified tRNA and tRNA synthetases to add to or substitute amino acidsthat are not encoded in naturally-occurring systems.

Methods for generating tRNAs and tRNA synthetases which use amino acidsthat are not encoded in naturally-occurring systems are described in,e.g., U.S. Patent Application Publications 2003/0082575 (Ser. No.10/126,927) and 2003/0108885 (Ser. No. 10/126,931) which areincorporated by reference herein. These methods involve generating atranslational machinery that functions independently of the synthetasesand tRNAs endogenous to the translation system (and are thereforesometimes referred to as “orthogonal”). Typically, the translationsystem comprises an orthogonal tRNA (O-tRNA) and an orthogonal aminoacyltRNA synthetase (O-RS). Typically, the O-RS preferentially aminoacylatesthe O-tRNA with at least one non-naturally occurring amino acid in thetranslation system and the O-tRNA recognizes at least one selector codonthat is not recognized by other tRNAs in the system. The translationsystem thus inserts the non-naturally-encoded amino acid into a proteinproduced in the system, in response to an encoded selector codon,thereby “substituting” an amino acid into a position in the encodedpolypeptide.

A wide variety of orthogonal tRNAs and aminoacyl tRNA synthetases havebeen described in the art for inserting particular synthetic amino acidsinto polypeptides, and are generally suitable for use in the presentinvention. For example, keto-specific O-tRNA/aminoacyl-tRNA synthetasesare described in Wang, L., et al., Proc. Natl. Acad. Sci. USA 100:56-61(2003) and Zhang, Z. et al., Biochem. 42(22):6735-6746 (2003). ExemplaryO-RS, or portions thereof, are encoded by polynucleotide sequences andinclude amino acid sequences disclosed in U.S. Patent ApplicationPublications 2003/0082575 and 2003/0108885, each incorporated herein byreference. Corresponding O-tRNA molecules for use with the O-RSs arealso described in U.S. Patent Application Publications 2003/0082575(Ser. No. 10/126,927) and 2003/0108885 (Ser. No. 10/126,931) which areincorporated by reference herein.

An example of an azide-specific O-tRNA/aminoacyl-tRNA synthetase systemis described in Chin, J. W., et al., J. Am. Chem. Soc. 124:9026-9027(2002). Exemplary O-RS sequences for p-azido-L-Phe include, but are notlimited to, nucleotide sequences SEQ ID NOs: 14-16 and 29-32 and aminoacid sequences SEQ ID NOs: 46-48 and 61-64 as disclosed in U.S. PatentApplication Publication 2003/0108885 (Ser. No. 10/126,931) which isincorporated by reference herein. Additional O-tRNA sequences include,but are not limited to, nucleotide sequences SEQ ID NOs: 1-3 asdisclosed in U.S. Patent Application Publication 2003/0108885 (Ser. No.10/126,931) which is incorporated by reference herein. Other examples ofO-tRNA/aminoacyl-tRNA synthetase pairs specific to particularnon-naturally encoded amino acids are described in U.S. PatentApplication Publication 2003/0082575 (Ser. No. 10/126,927) which isincorporated by reference herein. O-RS and O-tRNA that incorporate bothketo- and azide-containing amino acids in S. cerevisiae are described inChin, J. W., et al., Science 301:964-967 (2003).

Several other orthogonal pairs have been reported. Glutaminyl (see,e.g., Liu, D. R., and Schultz, P. G. (1999) Proc. Natl. Acad. Sci.U.S.A. 96:4780-4785), aspartyl (see, e.g., Pastrnak, M., et al., (2000)Helv. Chim. Acta 83:2277-2286), and tyrosyl (see, e.g., Ohno, S., etal., (1998) J. Biochem. (Tokyo, Jpn.) 124:1065-1068; and, Kowal, A. K.,et al., (2001) Proc. Natl. Acad. Sci. U.S.A. 98:2268-2273) systemsderived from S. cerevisiae tRNA's and synthetases have been describedfor the potential incorporation of unnatural amino acids in E. coli.Systems derived from the E. coli glutaminyl (see, e.g., Kowal, A. K., etal., (2001) Proc. Natl. Acad. Sci. U.S.A. 98:2268-2273) and tyrosyl(see, e.g., Edwards, H., and Schimmel, P. (1990) Mol. Cell. Biol.10:1633-1641) synthetases have been described for use in S. cerevisiae.The E. coli tyrosyl system has been used for the incorporation of3-iodo-L-tyrosine in vivo, in mammalian cells. See, Sakamoto, K., etal., (2002) Nucleic Acids Res. 30:4692-4699.

Use of O-tRNA/aminoacyl-tRNA synthetases involves selection of aspecific codon which encodes the non-naturally encoded amino acid. Whileany codon can be used, it is generally desirable to select a codon thatis rarely or never used in the cell in which the O-tRNA/aminoacyl-tRNAsynthetase is expressed. For example, exemplary codons include nonsensecodon such as stop codons (amber, ochre, and opal), four or more basecodons and other natural three-base codons that are rarely or unused.

Specific selector codon(s) can be introduced into appropriate positionsin the hGH polynucleotide coding sequence using mutagenesis methodsknown in the art (including but not limited to, site-specificmutagenesis, cassette mutagenesis, restriction selection mutagenesis,etc.).

Methods for generating components of the protein biosynthetic machinery,such as O-RSs, O-tRNAs, and orthogonal O-tRNA/O-RS pairs that can beused to incorporate a non-naturally encoded amino acid are described inWang, L., et al., Science 292: 498-500 (2001); Chin, J. W., et al., J.Am. Chem. Soc. 124:9026-9027 (2002); Zhang, Z. et al., Biochemistry 42:6735-6746 (2003). Methods and compositions for the in vivo incorporationof non-naturally encoded amino acids are described in U.S. PatentApplication Publication 2003/0082575 (Ser. No. 10/126,927) which isincorporated by reference herein. Methods for selecting an orthogonaltRNA-tRNA synthetase pair for use in in vivo translation system of anorganism are also described in U.S. Patent Application Publications2003/0082575 (Ser. No. 10/126,927) and 2003/0108885 (Ser. No.10/126,931) which are incorporated by reference herein. PCT PublicationNo. WO 04/035743 entitled “Site Specific Incorporation of Keto AminoAcids into Proteins,” which is incorporated by reference herein in itsentirety, describes orthogonal RS and tRNA pairs for the incorporationof keto amino acids. PCT Publication No. WO 04/094593 entitled“Expanding the Eukaryotic Genetic Code,” which is incorporated byreference herein in its entirety, describes orthogonal RS and tRNA pairsfor the incorporation of non-naturally encoded amino acids in eukaryotichost cells. Such methods are also detailed in U.S. patent applicationSer. No. 11/046,432 entitled “Modified Human Growth Hormone Polypeptidesand Their Uses,” which is incorporated by reference herein.

The organisms used in methods generating orthogonal tRNA and RS pairscomprise a variety of organisms and a variety of combinations. Forexample, the first and the second organisms of the methods can be thesame or different. In one embodiment, the organisms are optionally aprokaryotic organism, including but not limited to, Methanococcusjannaschii, Methanobacterium thermoautotrophicum, Halobacterium,Escherichia coli, A. fulgidus, P. furiosus, P. horikoshii, A. pernix, T.thermophilus, or the like. Alternatively, the organisms optionallycomprise a eukaryotic organism, including but not limited to, plants(including but not limited to, complex plants such as monocots, ordicots), algae, protists, fungi (including but not limited to, yeast,etc), animals (including but not limited to, mammals, insects,arthropods, etc.), or the like. In another embodiment, the secondorganism is a prokaryotic organism, including but not limited to,Methanococcus jannaschii, Methanobacterium thermoautotrophicum,Halobacterium, Escherichia coli, A. fulgidus, Halobacterium, P.furiosus, P. horikoshii, A. pernix, T. thermophilus, or the like.Alternatively, the second organism can be a eukaryotic organism,including but not limited to, a yeast, a animal cell, a plant cell, afungus, a mammalian cell, or the like. In various embodiments the firstand second organisms are different.

V. Location of Non-Naturally-Occurring Amino Acids in hGH Polypeptides

The present invention contemplates incorporation of one or morenon-naturally-occurring amino acids into hGH polypeptides. One or morenon-naturally-occurring amino acids may be incorporated at a particularposition which does not disrupt activity of the polypeptide. This can beachieved by making “conservative” substitutions, including but notlimited to, substituting hydrophobic amino acids with hydrophobic aminoacids, bulky amino acids for bulky amino acids, hydrophilic amino acidsfor hydrophilic amino acids, and/or inserting thenon-naturally-occurring amino acid in a location that is not requiredfor activity.

Regions of hGH can be illustrated as follows, wherein the amino acidpositions in hGH are indicated in the middle row (SEQ ID NO: 2 of U.S.patent application Ser. No. 11/046,432, which is incorporated byreference herein):

A variety of biochemical and structural approaches can be employed toselect the desired sites for substitution with a non-naturally encodedamino acid within the hGH polypeptide. It is readily apparent to thoseof ordinary skill in the art that any position of the polypeptide chainis suitable for selection to incorporate a non-naturally encoded aminoacid, and selection may be based on rational design or by randomselection for any or no particular desired purpose. Selection of desiredsites may be for producing a hGH molecule having any desired property oractivity, including but not limited to, agonists, super-agonists,inverse agonists, antagonists, receptor binding modulators, receptoractivity modulators, dimer or multimer formation, no change to activityor property compared to the native molecule, or manipulating anyphysical or chemical property of the polypeptide such as solubility,aggregation, or stability. For example, locations in the polypeptiderequired for biological activity of hGH polypeptides can be identifiedusing point mutation analysis, alanine scanning or homolog scanningmethods known in the art. See, e.g., Cunningham, B. and Wells, J.,Science, 244:1081-1085 (1989) (identifying 14 residues that are criticalfor hGH bioactivity) and Cunningham, B., et al. Science 243: 1330-1336(1989) (identifying antibody and receptor epitopes using homologscanning mutagenesis). U.S. Pat. Nos. 5,580,723; 5,834,250; 6,013,478;6,428,954; and 6,451,561, which are incorporated by reference herein,describe methods for the systematic analysis of the structure andfunction of polypeptides such as hGH by identifying active domains whichinfluence the activity of the polypeptide with a target substance.Residues other than those identified as critical to biological activityby alanine or homolog scanning mutagenesis may be good candidates forsubstitution with a non-naturally encoded amino acid depending on thedesired activity sought for the polypeptide. Alternatively, the sitesidentified as critical to biological activity may also be goodcandidates for substitution with a non-naturally encoded amino acid,again depending on the desired activity sought for the polypeptide.Another alternative would be to simply make serial substitutions in eachposition on the polypeptide chain with a non-naturally encoded aminoacid and observe the effect on the activities of the polypeptide. It isreadily apparent to those of ordinary skill in the art that any means,technique, or method for selecting a position for substitution with anon-natural amino acid into any polypeptide is suitable for use in thepresent invention.

The structure and activity of naturally-occurring mutants of hGHpolypeptides that contain deletions can also be examined to determineregions of the protein that are likely to be tolerant of substitutionwith a non-naturally encoded amino acid. See, e.g., Kostyo et al.,Biochem. Biophys. Acta, 925: 314 (1987); Lewis, U., et al., J. Biol.Chem., 253:2679-2687 (1978) for hGH. In a similar manner, proteasedigestion and monoclonal antibodies can be used to identify regions ofhGH that are responsible for binding the hGH receptor. See, e.g.,Cunningham, B., et al. Science 243: 1330-1336 (1989); Mills, J., et al.,Endocrinology, 107:391-399 (1980); Li, C., Mol. Cell. Biochem., 46:31-41(1982) (indicating that amino acids between residues 134-149 can bedeleted without a loss of activity). Once residues that are likely to beintolerant to substitution with non-naturally encoded amino acids havebeen eliminated, the impact of proposed substitutions at each of theremaining positions can be examined from the three-dimensional crystalstructure of the hGH and its binding proteins. See de Vos, A., et al.,Science, 255:306-312 (1992) for hGH; all crystal structures of hGH areavailable in the Protein Data Bank (including 3HHR, 1AXI, and 1HWG)(PDB, available on the World Wide Web at rcsb.org), a centralizeddatabase containing three-dimensional structural data of large moleculesof proteins and nucleic acids. Thus, those of skill in the art canreadily identify amino acid positions that can be substituted withnon-naturally encoded amino acids.

In some embodiments, the hGH polypeptides of the invention comprise oneor more non-naturally occurring amino acids positioned in a region ofthe protein that does not disrupt the helices or beta sheet secondarystructure of the polypeptide.

Exemplary residues of incorporation of a non-naturally encoded aminoacid may be those that are excluded from potential receptor bindingregions (including but not limited to, Site I and Site II), may be fullyor partially solvent exposed, have minimal or no hydrogen-bondinginteractions with nearby residues, may be minimally exposed to nearbyreactive residues, and may be in regions that are highly flexible(including but not limited to, C-D loop) or structurally rigid(including but not limited to, B helix) as predicted by thethree-dimensional crystal structure, secondary, tertiary or quaternarystructure of the hGH polypeptide bound or unbound to its receptor.

In some embodiments, one or more non-naturally encoded amino acids areincorporated at any position in one or more of the following regionscorresponding to secondary structures in hGH as follows: positionscorresponding to 1-5 (N-terminus), 6-33 (A helix), 34-74 (region betweenA helix and B helix, the A-B loop), 75-96 (B helix), 97-105 (regionbetween B helix and C helix, the B-C loop), 106-129 (C helix), 130-153(region between C helix and D helix, the C-D loop), 154-183 (D helix),184-191 (C-terminus) from SEQ ID NO: 2. In other embodiments, GHpolypeptides, e.g., hGH polypeptides of the invention comprise at leastone non-naturally-occurring amino acid substituted for at least oneamino acid located in at least one region of GH, e.g., hGH selected fromthe group consisting regions corresponding to the N-terminus (1-5), theN-terminal end of the A-B loop (32-46); the B-C loop (97-105), the C-Dloop (132-149), and the C-terminus (184-191) of SEQ ID NO: 2. In someembodiments, one or more non-naturally encoded amino acids areincorporated at one or more of the following positions of GH, e.g., hGHcorresponding to: before position 1 (i.e. at the N-terminus), 1, 2, 3,4, 5, 8, 9, 11, 12, 15, 16, 19, 22, 29, 30, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 52, 55, 57, 59, 65, 66, 69,70, 71, 74, 88, 91, 92, 94, 95, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 111, 112, 113, 115, 116, 119, 120, 122, 123,126, 127, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,155, 156, 158, 159, 161, 168, 172, 183, 184, 185, 186, 187, 188, 189,190, 191, 192 (i.e., at the carboxyl terminus of the protein) of SEQ IDNO: 2 or the corresponding amino acids of SEQ ID NO: 1 or 3.

Exemplary sites of incorporation of one or more non-naturally encodedamino acids include sites corresponding to 29, 30, 33, 34, 35, 37, 39,40, 49, 57, 59, 66, 69, 70, 71, 74, 88, 91, 92, 94, 95, 98, 99, 101,103, 107, 108, 111, 122, 126, 129, 130, 131, 133, 134, 135, 136, 137,139, 140, 141, 142, 143, 145, 147, 154, 155, 156, 159, 183, 186, and187, or any combination thereof from SEQ ID NO: 2 or the correspondingamino acids of SEQ ID NO: 1 or 3.

A subset of exemplary sites for incorporation of one or morenon-naturally encoded amino acid include sites corresponding to 29, 33,35, 37, 39, 49, 57, 69, 70, 71, 74, 88, 91, 92, 94, 95, 98, 99, 101,103, 107, 108, 111, 129, 130, 131, 133, 134, 135, 136, 137, 139, 140,141, 142, 143, 145, 147, 154, 155, 156, 186, and 187, or any combinationthereof from SEQ ID NO: 2 or the corresponding amino acids of SEQ ID NO:1 or 3. An examination of the crystal structure of GH, e.g., hGH and itsinteractions with the GH, e.g., hGH receptor indicates that the sidechains of these amino acid residues are fully or partially accessible tosolvent and the side chain of a non-naturally encoded amino acid maypoint away from the protein surface and out into the solvent.

Exemplary positions for incorporation of one or more non-naturallyencoded amino acids include sites corresponding to 35, 88, 91, 92, 94,95, 99, 101, 103, 111, 131, 133, 134, 135, 136, 139, 140, 143, 145, and155, or any combination thereof from SEQ ID NO: 2 or the correspondingamino acids of SEQ ID NO: 1 or 3. An examination of the crystalstructure of GH, e.g., hGH and its interactions with the GH, e.g., hGHreceptor indicates that the side chains of these amino acid residues arefully exposed to the solvent and the side chain of the native residuepoints out into the solvent.

A subset of exemplary sites for incorporation of one or morenon-naturally encoded amino acids include sites corresponding to 30, 74,103, or any combination thereof, from SEQ ID NO: 2 or the correspondingamino acids of SEQ ID NO: 1 or 3. Another subset of exemplary sites forincorporation of one or more non-naturally encoded amino acids includesites corresponding to 35, 92, 143, 145, or any combination thereof,from SEQ ID NO: 2 or the corresponding amino acids of SEQ ID NO: 1 or 3.A further subset of exemplary sites for incorporation of one or morenon-naturally encoded amino acids include sites corresponding to 35, 92,131, 134, 143, 145, or any combination thereof, from SEQ ID NO: 2 or thecorresponding amino acids of SEQ ID NO: 1 or 3. Still a further subsetof exemplary sites for incorporation of one or more non-naturallyencoded amino acids include sites corresponding to 30, 35, 74, 92, 103,145, or any combination thereof, from SEQ ID NO: 2 or the correspondingamino acids of SEQ ID NO: 1 or 3. Yet a further subset of exemplarysites for incorporation of one or more non-naturally encoded amino acidsinclude sites corresponding to 35, 92, 143, 145, or any combinationthereof, from SEQ ID NO: 2 or the corresponding amino acids of SEQ IDNO: 1 or 3. In certain embodiments, sites for incorporation of one ormore non-naturally encoded amino acids include a site corresponding to35 from SEQ ID NO: 2 or the corresponding amino acids of SEQ ID NO: 1 or3.

In some embodiments, at least one of the non-naturally encoded aminoacids incorporated into the GH, e.g., hGH, contains a carbonyl group,e.g., a ketone group. In certain embodiments, at least one of thenon-naturally encoded amino acids incorporated into the GH, e.g., hGH ispara-acetylphenylalanine. In some embodiments in which the GH, e.g., hGHcontains a plurality of non-naturally-encoded amino acids, more than oneof the non-naturally-encoded amino acids incorporated into the GH, e.g.,hGH is para-acetylphenylalanine. In some embodiments in which the GH,e.g., hGH contains a plurality of non-naturally-encoded amino acids,substantially all of the non-naturally-encoded amino acids incorporatedinto the GH, e.g., hGH are para-acetylphenylalanine.

In some embodiments, the non-naturally occurring amino acid is linked toa water soluble polymer at one or more positions, including but notlimited to, positions corresponding to: before position 1 (i.e. at theN-terminus), 1, 2, 3, 4, 5, 8, 9, 11, 12, 15, 16, 19, 22, 29, 30, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 52,55, 57, 59, 65, 66, 69, 70, 71, 74, 88, 91, 92, 94, 95, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 111, 112, 113, 115, 116,119, 120, 122, 123, 126, 127, 129, 130, 131, 132, 133, 134, 135, 136,137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150,151, 152, 153, 154, 155, 156, 158, 159, 161, 168, 172, 183, 184, 185,186, 187, 188, 189, 190, 191, 192 (i.e., at the carboxyl terminus of theprotein) (SEQ ID NO: 2 or the corresponding amino acids of SEQ ID NO: 1or 3). In some embodiments, the non-naturally occurring amino acid islinked to a water soluble polymer at positions including but not limitedto, positions corresponding to one or more of these positions: 30, 35,74, 92, 103, 143, 145 (SEQ ID NO: 2 or the corresponding amino acids ofSEQ ID NO: 1 or 3). In some embodiments, the non-naturally occurringamino acid is linked to a water soluble polymer at positions includingbut not limited to, positions corresponding to one or more of thesepositions: 35, 92, 143, 145 (SEQ ID NO: 2 or the corresponding aminoacids of SEQ ID NO: 1 or 3). In some embodiments, the non-naturallyoccurring amino acid is linked to a water soluble polymer at positionsincluding but not limited to, positions corresponding to one or more ofthese positions: 35, 92, 131, 134, 143, 145, or any combination thereof,from SEQ ID NO: 2 or the corresponding amino acids of SEQ ID NO: 1 or 3.In some embodiments, the non-naturally occurring amino acid is linked toa water soluble polymer at positions including but not limited to,positions corresponding to one or more of these positions: 30, 35, 74,92, 103, 145, or any combination thereof, from SEQ ID NO: 2 or thecorresponding amino acids of SEQ ID NO: 1 or 3. In some embodiments, thenon-naturally occurring amino acid is linked to a water soluble polymerat positions including but not limited to, positions corresponding toone or more of these positions: 35, 92, 143, 145, or any combinationthereof, from SEQ ID NO: 2 or the corresponding amino acids of SEQ IDNO: 1 or 3. In some embodiments, the non-naturally occurring amino acidis linked to a water-soluble polymer at a position corresponding to, butnot limited to, position 35 from SEQ ID NO: 2 or the corresponding aminoacids of SEQ ID NO: 1 or 3 is linked to a water-soluble polymer.

In some embodiments the water-soluble polymer(s) linked to the GH, e.g.,hGH, include one or more polyethylene glycol molecules (PEGs). Thepolymer, e.g., PEG, may be linear or branched. Typically, linearpolymers, e.g., PEGs, used in the invention can have a MW of about 0.1to about 100 kDa, or about 1 to about 60 kDa, or about 20 to about 40kDa, or about 30 kDa. Typically, branched polymers, e.g., PEGs, used inthe invention can have a MW of about 1 to about 100 kDa, or about 30 toabout 50 kDa, or about 40 kDa. Polymers such as PEGs are describedfurther herein. In certain embodiments, the linkage between the GH,e.g., hGH and the water-soluble polymer, e.g., PEG, is an oxime bond.

Certain embodiments of the invention encompass compositions that includea GH, e.g., hGH, linked to at least one water-soluble polymer by acovalent bond, where the covalent bond is an oxime bond. In someembodiments, the water-soluble polymer is a PEG, e.g., a linear PEG. Insome embodiments encompassing at least one linear PEG linked by an oximebond to a GH, e.g., hGH, the PEG can have a MW of about 0.1 to about 100kDa, or about 1 to about 60 kDa, or about 20 to about 40 kDa, or about30 kDa. In certain embodiments encompassing a linear PEG linked by anoxime bond to a GH, e.g., hGH, the PEG has a MW of about 30 kDa. In someembodiments encompassing at least one branched PEG linked by an oximebond to a GH, e.g., hGH, the PEG can have a MW of about 1 to about 100kDa or about 30 to about 50 kDa, or about 40 kDa. In certain embodimentsencompassing a branched PEG linked by an oxime bond to a GH, e.g., hGH,the PEG has a MW of about 40 kDa. In some embodiments, the GH is a GH,e.g., hGH and in certain of these embodiments, the GH, e.g., hGH has asequence that is at least about 80% identical to SEQ ID NO: 2; in someembodiments the GH, e.g., hGH has a sequence that is the sequence of SEQID NO: 2. In some embodiments, the GH, e.g., hGH, contains at least onenon-naturally-encoded amino acid; in some of these embodiments, at leastone oxime bond is between the non-naturally-encoded amino acid and atleast one water-soluble polymer. In some embodiments, thenon-naturally-encoded amino acid contains a carbonyl group, such as aketone group; in some embodiments, the non-naturally-encoded amino acidis para-acetylphenylalanine. In some embodiments, thepara-acetylphenylalanine is substituted at a position corresponding toposition 35 of SEQ ID NO: 2.

Thus, in some embodiments, the invention provides a GH, e.g., hGH,linked to at least one water-soluble polymer, e.g., a PEG, by a covalentbond, where the covalent bond is an oxime bond. In certain embodiments,the water-soluble polymer is a PEG and the PEG is a linear PEG. In theseembodiments, the linear PEG has a MW of about 0.1 to about 100 kDa, orabout 1 to about 60 kDa, or about 20 to about 40 kDa, or about 30 kDa.In certain embodiments encompassing a linear PEG linked by an oxime bondto a GH, e.g., hGH, the PEG has a MW of about 30 kDa. In certainembodiments, the water-soluble polymer is a PEG that is a branched PEG.In these embodiments, the branched PEG has a MW of about 1 to about 100kDa, or about 30 to about 50 kDa, or about 40 kDa. In certainembodiments encompassing a branched PEG linked by an oxime bond to a GH,e.g., hGH, the PEG has a MW of about 40 kDa.

In some embodiments, the invention provides a GH, e.g., hGH, where theGH, e.g., hGH contains a non-naturally encoded amino acid, where the GHis linked to at least one water-soluble polymer, e.g., a PEG, by acovalent bond, and where the covalent bond is an oxime bond between thenon-naturally encoded amino acid and the water-soluble polymer, e.g.,PEG. In some embodiments, the non-naturally-encoded amino acid isincorporated into the GH, e.g., hGH, at a position corresponding toposition 35 of SEQ ID NO: 2. In certain embodiments where thewater-soluble polymer is a PEG, the PEG is a linear PEG. In theseembodiments, the linear PEG has a MW of about 0.1 to about 100 kDa, orabout 1 to about 60 kDa, or about 20 to about 40 kDa, or about 30 kDa.In certain embodiments encompassing a linear PEG linked by an oxime bondto a GH, e.g., hGH, the PEG has a MW of about 30 kDa. In certainembodiments where the water-soluble polymer is a PEG, the PEG is abranched PEG. In these embodiments, the branched PEG has a MW of about 1to about 100 kDa, or about 30 to about 50 kDa, or about 40 kDa. Incertain embodiments encompassing a branched PEG linked by an oxime bondto a GH, e.g., hGH, the PEG has a MW of about 40 kDa.

In some embodiments, the invention provides a GH, e.g., hGH, where theGH, e.g., hGH contains a non-naturally encoded amino acid that is acarbonyl-containing non-naturally encoded amino acid, where the GH islinked to at least one water-soluble polymer, e.g., a PEG, by a covalentbond, and where the covalent bond is an oxime bond between thenon-naturally encoded carbonyl-containing amino acid and thewater-soluble polymer, e.g., PEG. In some embodiments, thenon-naturally-encoded carbonyl-containing amino acid is incorporatedinto the GH, e.g., hGH, at a position corresponding to position 35 ofSEQ ID NO: 2. In certain embodiments where the water-soluble polymer isa PEG, the PEG is a linear PEG. In these embodiments, the linear PEG hasa MW of about 0.1 to about 100 kDa, or about 1 to about 60 kDa, or about20 to about 40 kDa, or about 30 kDa. In certain embodiments encompassinga linear PEG linked by an oxime bond to a GH, e.g., hGH, the PEG has aMW of about 30 kDa. In certain embodiments where the water-solublepolymer is a PEG, the PEG is a branched PEG. In these embodiments, thebranched PEG has a MW of about 1 to about 100 kDa, or about 30 to about50 kDa, or about 40 kDa. In certain embodiments encompassing a branchedPEG linked by an oxime bond to a GH, e.g., hGH, the PEG has a MW ofabout 40 kDa.

In some embodiments, the invention provides a GH, e.g., hGH, thatcontains a non-naturally encoded amino acid that includes a ketonegroup, where the GH is linked to at least one water-soluble polymer,e.g., a PEG, by a covalent bond, and where the covalent bond is an oximebond between the non-naturally encoded amino acid containing a ketonegroup and the water-soluble polymer, e.g., PEG. In some embodiments, thenon-naturally-encoded amino acid containing a ketone group isincorporated into the GH, e.g., hGH, at a position corresponding toposition 35 of SEQ ID NO: 2. In certain embodiments where thewater-soluble polymer is a PEG, the PEG is a linear PEG. In theseembodiments, the linear PEG has a MW of about 0.1 to about 100 kDa, orabout 1 to about 60 kDa, or about 20 to about 40 kDa, or about 30 kDa.In certain embodiments encompassing a linear PEG linked by an oxime bondto a GH, e.g., hGH, the PEG has a MW of about 30 kDa. In certainembodiments where the water-soluble polymer is a PEG, the PEG is abranched PEG. In these embodiments, the branched PEG has a MW of about 1to about 100 kDa, or about 30 to about 50 kDa, or about 40 kDa. Incertain embodiments encompassing a branched PEG linked by an oxime bondto a GH, e.g., hGH, the PEG has a MW of about 40 kDa.

In some embodiments, the invention provides a GH, e.g., hGH, thatcontains a non-naturally encoded amino acid that is apara-acetylphenylalanine, where the GH linked to at least onewater-soluble polymer, e.g., a PEG, by a covalent bond, and where thecovalent bond is an oxime bond between the para-acetylphenylalanine andthe water-soluble polymer, e.g., PEG. In some embodiments, thepara-acetylphenylalanine is incorporated into the GH, e.g., hGH, at aposition corresponding to position 35 of SEQ ID NO: 2. In certainembodiments where the water-soluble polymer is a PEG, the PEG is alinear PEG. In these embodiments, the linear PEG has a MW of about 0.1to about 100 kDa, or about 1 to about 60 kDa, or about 20 to about 40kDa, or about 30 kDa. In certain embodiments encompassing a linear PEGlinked by an oxime bond to a GH, e.g., hGH, the PEG has a MW of about 30kDa. In certain embodiments where the water-soluble polymer is a PEG,the PEG is a branched PEG. In these embodiments, the branched PEG has aMW of about 1 to about 100 kDa, or about 30 to about 50 kDa, or about 40kDa. In certain embodiments encompassing a branched PEG linked by anoxime bond to a GH, e.g., hGH, the PEG has a MW of about 40 kDa.

In certain embodiments the invention provides a GH, e.g., hGH thatincludes SEQ ID NO: 2, and where the GH, e.g., hGH is substituted at aposition corresponding to position 35 of SEQ ID NO: 2 with apara-acetylphenylalanine that is linked by an oxime linkage to a linearPEG of MW of about 30 kDa.

In some embodiments, the invention provides a hormone composition thatincludes a GH, e.g., hGH, linked via an oxime bond to at least one PEG,e.g., a linear PEG, where the GH, e.g., hGH comprises the amino acidsequence of SEQ ID NO: 2, and where the GH, e.g., hGH contains at leastone non-naturally-encoded amino acid substituted at one or morepositions including, but not limited to, positions corresponding to:before position 1 (i.e. at the N-terminus), 1, 2, 3, 4, 5, 8, 9, 11, 12,15, 16, 19, 22, 29, 30, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 52, 55, 57, 59, 65, 66, 69, 70, 71, 74, 88, 91,92, 94, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,109, 111, 112, 113, 115, 116, 119, 120, 122, 123, 126, 127, 129, 130,131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144,145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 158, 159,161, 168, 172, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192 (i.e.,at the carboxyl terminus of the protein) (SEQ ID NO: 2 or thecorresponding amino acids of SEQ ID NO: 1 or 3). In some embodiments,the invention provides a hormone composition that includes a GH, e.g.,hGH, linked via an oxime bond to at least one PEG, e.g., a linear PEG,where the GH, e.g., hGH comprises the amino acid sequence of SEQ ID NO:2, and where the GH, e.g., hGH contains at least onenon-naturally-encoded amino acid substituted at one or more positionsincluding, but not limited to, positions corresponding to: 30, 35, 74,92, 103, 143, 145 (SEQ ID NO: 2 or the corresponding amino acids of SEQID NO: 1 or 3). In some embodiments, the invention provides a hormonecomposition that includes a GH, e.g., hGH, linked via an oxime bond toat least one PEG, e.g., a linear PEG, where the GH, e.g., hGH comprisesthe amino acid sequence of SEQ ID NO: 2, and where the GH, e.g., hGHcontains at least one non-naturally-encoded amino acid substituted atone or more positions including, but not limited to, positionscorresponding to: 35, 92, 143, 145 (SEQ ID NO: 2 or the correspondingamino acids of SEQ ID NO: 1 or 3). In some embodiments, the inventionprovides a hormone composition that includes a GH, e.g., hGH, linked viaan oxime bond to at least one PEG, e.g., a linear PEG, where the GH,e.g., hGH comprises the amino acid sequence of SEQ ID NO: 2, and wherethe GH, e.g., hGH contains at least one non-naturally-encoded amino acidsubstituted at one or more positions including, but not limited to,positions corresponding to: 35, 92, 131, 134, 143, 145, or anycombination thereof, from SEQ ID NO: 2 or the corresponding amino acidsof SEQ ID NO: 1 or 3. In some embodiments, the invention provides ahormone composition that includes a GH, e.g., hGH, linked via an oximebond to at least one PEG, e.g., a linear PEG, where the GH, e.g., hGHcomprises the amino acid sequence of SEQ ID NO: 2, and where the GH,e.g., hGH contains at least one non-naturally-encoded amino acidsubstituted at one or more positions including, but not limited to,positions corresponding to: 30, 35, 74, 92, 103, 145, or any combinationthereof, from SEQ ID NO: 2 or the corresponding amino acids of SEQ IDNO: 1 or 3. In some embodiments, the invention provides a hormonecomposition that includes a GH, e.g., hGH, linked via an oxime bond toat least one PEG, e.g., a linear PEG, where the GH, e.g., hGH comprisesthe amino acid sequence of SEQ ID NO: 2, and where the GH, e.g., hGHcontains at least one non-naturally-encoded amino acid substituted atone or more positions including, but not limited to, positionscorresponding to: 35, 92, 143, 145, or any combination thereof, from SEQID NO: 2 or the corresponding amino acids of SEQ ID NO: 1 or 3. In someembodiments, the invention provides a hormone composition that includesa GH, e.g., hGH, linked via an oxime bond to at least one PEG, e.g., alinear PEG, where the GH, e.g., hGH comprises the amino acid sequence ofSEQ ID NO: 2, and where the GH, e.g., hGH contains at least onenon-naturally-encoded amino acid substituted at one or more positionsincluding, but not limited to, positions corresponding to position 35from SEQ ID NO: 2 or the corresponding amino acids of SEQ ID NO: 1 or 3.In embodiments in which the PEG is a linear PEG, the PEG can have a MWof about 0.1 to about 100 kDa, or about 1 to about 60 kDa, or about 20to about 40 kDa, or about 30 kDa.

In some embodiments, the invention provides a hormone composition thatincludes a GH, e.g., hGH, linked via an oxime bond to at least one PEG,e.g., a linear PEG, where the GH, e.g., hGH includes the amino acidsequence of SEQ ID NO: 2, and where the GH, e.g., hGH contains at leastone non-naturally-encoded amino acid that is a para-acetylphenylalaninesubstituted at one or more positions including, but not limited to,positions corresponding to: before position 1 (i.e. at the N-terminus),1, 2, 3, 4, 5, 8, 9, 11, 12, 15, 16, 19, 22, 29, 30, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 52, 55, 57, 59, 65,66, 69, 70, 71, 74, 88, 91, 92, 94, 95, 97, 98, 99, 100, 101, 102, 103,104, 105, 106, 107, 108, 109, 111, 112, 113, 115, 116, 119, 120, 122,123, 126, 127, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139,140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153,154, 155, 156, 158, 159, 161, 168, 172, 183, 184, 185, 186, 187, 188,189, 190, 191, 192 (i.e., at the carboxyl terminus of the protein) (SEQID NO: 2 or the corresponding amino acids of SEQ ID NO: 1 or 3). In someembodiments, the invention provides a hormone composition that includesa GH, e.g., hGH, linked via an oxime bond to at least one PEG, e.g., alinear PEG, where the GH, e.g., hGH comprises the amino acid sequence ofSEQ ID NO: 2, and where the GH, e.g., hGH contains at least onenon-naturally-encoded amino acid that is a para-acetylphenylalaninesubstituted at one or more positions including, but not limited to,positions corresponding to: 30, 35, 74, 92, 103, 143, 145 (SEQ ID NO: 2or the corresponding amino acids of SEQ ID NO: 1 or 3). In someembodiments, the invention provides a hormone composition that includesa GH, e.g., hGH, linked via an oxime bond to at least one PEG, e.g., alinear PEG, where the GH, e.g., hGH comprises the amino acid sequence ofSEQ ID NO: 2, and where the GH, e.g., hGH contains at least onenon-naturally-encoded amino acid that is a para-acetylphenylalaninesubstituted at one or more positions including, but not limited to,positions corresponding to: 35, 92, 143, 145 (SEQ ID NO: 2 or thecorresponding amino acids of SEQ ID NO: 1 or 3). In some embodiments,the invention provides a hormone composition that includes a GH, e.g.,hGH, linked via an oxime bond to at least one PEG, e.g., a linear PEG,where the GH, e.g., hGH comprises the amino acid sequence of SEQ ID NO:2, and where the GH, e.g., hGH contains at least onenon-naturally-encoded amino acid that is a para-acetylphenylalaninesubstituted at one or more positions including, but not limited to,positions corresponding to: 35, 92, 131, 134, 143, 145, or anycombination thereof, from SEQ ID NO: 2 or the corresponding amino acidsof SEQ ID NO: 1 or 3. In some embodiments, the invention provides ahormone composition that includes a GH, e.g., hGH, linked via an oximebond to at least one PEG, e.g., a linear PEG, where the GH, e.g., hGHcomprises the amino acid sequence of SEQ ID NO: 2, and where the GH,e.g., hGH contains at least one non-naturally-encoded amino acid that isa para-acetylphenylalanine substituted at one or more positionsincluding, but not limited to, positions corresponding to: 30, 35, 74,92, 103, 145, or any combination thereof, from SEQ ID NO: 2 or thecorresponding amino acids of SEQ ID NO: 1 or 3. In some embodiments, theinvention provides a hormone composition that includes a GH, e.g., hGH,linked via an oxime bond to at least one PEG, e.g., a linear PEG, wherethe GH, e.g., hGH comprises the amino acid sequence of SEQ ID NO: 2, andwhere the GH, e.g., hGH contains at least one non-naturally-encodedamino acid that is a para-acetylphenylalanine substituted at one or morepositions including, but not limited to, positions corresponding to: 35,92, 143, 145, or any combination thereof, from SEQ ID NO: 2 or thecorresponding amino acids of SEQ ID NO: 1 or 3. In some embodiments, theinvention provides a hormone composition that includes a GH, e.g., hGH,linked via an oxime bond to at least one PEG, e.g., a linear PEG, wherethe GH, e.g., hGH comprises the amino acid sequence of SEQ ID NO: 2, andwhere the GH, e.g., hGH contains at least one non-naturally-encodedamino acid that is a para-acetylphenylalanine substituted at one or morepositions including, but not limited to, positions corresponding toposition 35 from SEQ ID NO: 2 or the corresponding amino acids of SEQ IDNO: 1 or 3. In embodiments in which the PEG is a linear PEG, the PEG canhave a MW of about 0.1 to about 100 kDa, or about 1 to about 60 kDa, orabout 20 to about 40 kDa, or about 30 kDa.

In some embodiments, the invention provides a GH, e.g., hGH, where theGH, e.g., hGH contains at least one non-naturally encoded amino acid,where the GH is linked to a plurality of water-soluble polymers, e.g., aplurality of PEGs, by covalent bonds, where one or more of the covalentbond is an oxime bond between at least one of the non-naturally encodedamino acid and the water-soluble polymer, e.g., PEG. The GH, e.g., hGH,may be linked to about 2-100 water-soluble polymers, e.g., PEGs, orabout 2-50 water-soluble polymers, e.g., PEGs, or about 2-25water-soluble polymers, e.g., PEGs, or about 2-10 water-solublepolymers, e.g., PEGs, or about 2-5 water-soluble polymers, e.g., PEGs,or about 5-100 water-soluble polymers, e.g., PEGs, or about 5-50water-soluble polymers, e.g., PEGs, or about 5-25 water-solublepolymers, e.g., PEGs, or about 5-10 water-soluble polymers, e.g., PEGs,or about 10-100 water-soluble polymers, e.g., PEGs, or about 10-50water-soluble polymers, e.g., PEGs, or about 10-20 water-solublepolymers, e.g., PEGs, or about 20-100 water-soluble polymers, e.g.,PEGs, or about 20-50 water-soluble polymers, e.g., PEGs, or about 50-100water-soluble polymers, e.g., PEGs. The one or morenon-naturally-encoded amino acids may be incorporated into the GH, e.g.,hGH, at any position described herein. In some embodiments, at least onenon-naturally-encoded amino acid is incorporated into the GH, e.g., hGH,at a position corresponding to position 35 of SEQ ID NO: 2. In someembodiments, the non-naturally encoded amino acids include at least onenon-naturally encoded amino acid that is a carbonyl-containingnon-naturally encoded amino acid, e.g., a ketone-containingnon-naturally encoded amino acid such as a para-acetylphenylalanine. Insome embodiments, the GH, e.g., hGH, includes apara-acetylphenylalanine. In some embodiments, thepara-acetylphenylalanine is incorporated into the GH, e.g., hGH, at aposition corresponding to position 35 of SEQ ID NO: 2, where thepara-acetylphenylalanine is linked to one of the polymers, e.g., one ofthe PEGs, by an oxime bond. In some embodiments, at least one of thewater-soluble polymers, e.g., PEGs, is linked to the GH, e.g., hGH, by acovalent bond to at least one of the non-naturally-encoded amino acids.In some embodiments, the covalent bond is an oxime bond. In someembodiments, a plurality of the water-soluble polymers, e.g., PEGs, arelinked to the GH, e.g., hGH, by covalent bonds to a plurality of thenon-naturally-encoded amino acids. In some embodiments, at least one thecovalent bonds is an oxime bond; in some embodiments, a plurality of thecovalent bonds are oxime bonds; in some embodiments, substantially allof the bonds are oxime bonds. The plurality of water-soluble polymers,e.g., PEG, may be linear, branched, or any combination thereof. Inembodiments that incorporate one or more linear PEGs, the linear PEGshave a MW of about 0.1 to about 100 kDa, or about 1 to about 60 kDa, orabout 20 to about 40 kDa, or about 30 kDa. In embodiments thatincorporate one or more branched PEGs, the branched PEGs have a MW ofabout 1 to about 100 kDa, or about 30 to about 50 kDa, or about 40 kDa.It will be appreciated that embodiments employing a plurality ofwater-soluble polymers, e.g., PEGs, will, in general, employ suchpolymers at lower MWs than embodiments in which a single PEG is used.Thus, in some embodiments, the overall MW of the plurality of PEGs isabout 0.1-500 kDa, or about 0.1-200 kDa, or about 0.1-100 kDa, or about1-1000 kDa, or about 1-500 kDa, or about 1-200 kDa, or about 1-100 kDa,or about 10-1000 kDa, or about 10-500 kDa, or about 10-200 kDa, or about10-100 kDa, or about 10-50 kDa, or about 20-1000 kDa, or about 20-500kDa, or about 20-200 kDa, or about 20-100 kDa, or about 20-80 kDa, about20-60 kDa, about 5-100 kDa, about 5-50 kDa, or about 5-20 kDa.

Human GH antagonists include, but are not limited to, those withsubstitutions at: 1, 2, 3, 4, 5, 8, 9, 11, 12, 15, 16, 19, 22, 103, 109,112, 113, 115, 116, 119, 120, 123, and 127 or an addition at position 1(i.e., at the N-terminus), or any combination thereof (SEQ ID NO:2, orthe corresponding amino acid in SEQ ID NO: 1, 3, or any other GHsequence).

A wide variety of non-naturally encoded amino acids can be substitutedfor, or incorporated into, a given position in a hGH polypeptide. Ingeneral, a particular non-naturally encoded amino acid is selected forincorporation based on an examination of the three dimensional crystalstructure of a hGH polypeptide with its receptor, a preference forconservative substitutions (i.e., aryl-based non-naturally encoded aminoacids, such as p-acetylphenylalanine or O-propargyltyrosine substitutingfor Phe, Tyr or Trp), and the specific conjugation chemistry that onedesires to introduce into the hGH polypeptide (e.g., the introduction of4-azidophenylalanine if one wants to effect a Huisgen [3+2]cycloadditionwith a water soluble polymer bearing an alkyne moiety or a amide bondformation with a water soluble polymer that bears an aryl ester that, inturn, incorporates a phosphine moiety).

In one embodiment, the method further includes incorporating into theprotein the unnatural amino acid, where the unnatural amino acidcomprises a first reactive group; and contacting the protein with amolecule (including but not limited to, a label, a dye, a polymer, awater-soluble polymer, a derivative of polyethylene glycol, aphotocrosslinker, a radionuclide, a cytotoxic compound, a drug, anaffinity label, a photoaffinity label, a reactive compound, a resin, asecond protein or polypeptide or polypeptide analog, an antibody orantibody fragment, a metal chelator, a cofactor, a fatty acid, acarbohydrate, a polynucleotide, a DNA, a RNA, an antisensepolynucleotide, a saccharide, a water-soluble dendrimer, a cyclodextrin,an inhibitory ribonucleic acid, a biomaterial, a nanoparticle, a spinlabel, a fluorophore, a metal-containing moiety, a radioactive moiety, anovel functional group, a group that covalently or noncovalentlyinteracts with other molecules, a photocaged moiety, an actinicradiation excitable moiety, a photoisomerizable moiety, biotin, aderivative of biotin, a biotin analogue, a moiety incorporating a heavyatom, a chemically cleavable group, a photocleavable group, an elongatedside chain, a carbon-linked sugar, a redox-active agent, an aminothioacid, a toxic moiety, an isotopically labeled moiety, a biophysicalprobe, a phosphorescent group, a chemiluminescent group, an electrondense group, a magnetic group, an intercalating group, a chromophore, anenergy transfer agent, a biologically active agent, a detectable label,a small molecule, a quantum dot, a nanotransmitter, a radionucleotide, aradiotransmitter, a neutron-capture agent, or any combination of theabove, or any other desirable compound or substance that comprises asecond reactive group.

In some cases, the non-naturally encoded amino acid substitution(s) willbe combined with other additions, substitutions or deletions within thehGH polypeptide to affect other biological traits of the hGHpolypeptide. In some cases, the other additions, substitutions ordeletions may increase the stability (including but not limited to,resistance to proteolytic degradation) of the hGH polypeptide orincrease affinity of the hGH polypeptide for its receptor. In somecases, the other additions, substitutions or deletions may increase thesolubility (including but not limited to, when expressed in E. coli orother host cells) of the hGH polypeptide. In some embodiments additions,substitutions or deletions may increase the polypeptide solubilityfollowing expression in E. coli or other recombinant host cells. In someembodiments sites are selected for substitution with a naturally encodedor non-natural amino acid in addition to another site for incorporationof a non-natural amino acid that results in increasing the polypeptidesolubility following expression in E. coli or other recombinant hostcells. In some embodiments, the hGH polypeptides comprise anotheraddition, substitution or deletion that modulates affinity for the hGHpolypeptide receptor, modulates (including but not limited to, increasesor decreases) receptor dimerization, stabilizes receptor dimers,modulates circulating half-life, modulates release or bio-availability,facilitates purification, or improves or alters a particular route ofadministration. A number of such alterations are described in U.S.patent application Ser. No. 11/046,432 entitled “Modified Human GrowthHormone Polypeptides and Their Uses,” which is incorporated by referenceherein in its entirety. Similarly, hGH polypeptides can compriseprotease cleavage sequences, reactive groups, antibody-binding domains(including but not limited to, FLAG or poly-His) or other affinity basedsequences (including, but not limited to, FLAG, poly-His, GST, etc.) orlinked molecules (including, but not limited to, biotin) that improvedetection (including, but not limited to, GFP), purification or othertraits of the polypeptide.

In some embodiments, the substitution of a non-naturally encoded aminoacid generates an GH, e.g., hGH antagonist. A subset of exemplary sitesfor incorporation of one or more non-naturally encoded amino acidinclude: 1, 2, 3, 4, 5, 8, 9, 11, 12, 15, 16, 19, 22, 103, 109, 112,113, 115, 116, 119, 120, 123, 127, or an addition before position 1 (SEQID NO: 2, or the corresponding amino acid in SEQ ID NO: 1, 3, or anyother GH sequence). In some embodiments, GH, e.g., hGH antagonistscomprise at least one substitution in the regions 1-5 (N-terminus), 6-33(A helix), 34-74 (region between A helix and B helix, the A-B loop),75-96 (B helix), 97-105 (region between B helix and C helix, the B-Cloop), 106-129 (C helix), 130-153 (region between C helix and D helix,the C-D loop), 154-183 (D helix), 184-191 (C-terminus) that cause GH toact as an antagonist. In other embodiments, the exemplary sites ofincorporation of a non-naturally encoded amino acid include residueswithin the amino terminal region of helix A and a portion of helix C. Inanother embodiment, substitution of G120 with a non-naturally encodedamino acid such as p-azido-L-phenyalanine or O-propargyl-L-tyrosine. Inother embodiments, the above-listed substitutions are combined withadditional substitutions that cause the GH, e.g., hGH polypeptide to bean GH, e.g., hGH antagonist. For instance, a non-naturally encoded aminoacid is substituted at one of the positions identified herein and asimultaneous substitution is introduced at G120 (e.g., G120R, G120K,G120W, G120Y, G120F, or G120E). In some embodiments, the GH, e.g., hGHantagonist comprises a non-naturally encoded amino acid linked to awater soluble polymer that is present in a receptor binding region ofthe GH, e.g., hGH molecule.

In some cases, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids aresubstituted with one or more non-naturally-encoded amino acids. In somecases, the GH, e.g., hGH polypeptide further includes 1, 2, 3, 4, 5, 6,7, 8, 9, 10, or more substitutions of one or more non-naturally encodedamino acids for naturally-occurring amino acids. For example, in someembodiments, one or more residues in the following regions of GH, e.g.,hGH are substituted with one or more non-naturally encoded amino acids:1-5 (N-terminus); 32-46 (N-terminal end of the A-B loop); 97-105 (B-Cloop); and 132-149 (C-D loop); and 184-191 (C-terminus). In someembodiments, one or more residues in the following regions of GH, e.g.,hGH are substituted with one or more non-naturally encoded amino acids:1-5 (N-terminus), 6-33 (A helix), 34-74 (region between A helix and Bhelix, the A-B loop), 75-96 (B helix), 97-105 (region between B helixand C helix, the B-C loop), 106-129 (C helix), 130-153 (region between Chelix and D helix, the C-D loop), 154-183 (D helix), 184-191(C-terminus). In some cases, the one or more non-naturally encodedresidues are linked to one or more lower molecular weight linear orbranched PEGs (approximately ˜5-20 kDa in mass or less), therebyenhancing binding affinity and comparable serum half-life relative tothe species attached to a single, higher molecular weight PEG.

In some embodiments, up to two of the following residues of GH, e.g.,hGH are substituted with one or more non-naturally-encoded amino acidsat position: 29, 30, 33, 34, 35, 37, 39, 40, 49, 57, 59, 66, 69, 70, 71,74, 88, 91, 92, 94, 95, 98, 99, 101, 103, 107, 108, 111, 122, 126, 129,130, 131, 133, 134, 135, 136, 137, 139, 140, 141, 142, 143, 145, 147,154, 155, 156, 159, 183, 186, and 187. In some cases, any of thefollowing pairs of substitutions are made: K38X* and K140X*; K41X* andK145X*; Y35X* and E88X*; Y35X* and F92X*; Y35X* and Y143X*; F92X* andY143X* wherein X* represents a non-naturally encoded amino acid.Preferred sites for incorporation of two or more non-naturally encodedamino acids include combinations of the following residues: 29, 33, 35,37, 39, 49, 57, 69, 70, 71, 74, 88, 91, 92, 94, 95, 98, 99, 101, 103,107, 108, 111, 129, 130, 131, 133, 134, 135, 136, 137, 139, 140, 141,142, 143, 145, 147, 154, 155, 156, 186, and 187. Particularly preferredsites for incorporation of two or more non-naturally encoded amino acidsinclude combinations of the following residues: 35, 88, 91, 92, 94, 95,99, 101, 103, 111, 131, 133, 134, 135, 136, 139, 140, 143, 145, and 155.

Preferred sites for incorporation in GH, e.g., hGH of two or morenon-naturally encoded amino acids include combinations of the followingresidues: before position 1 (i.e. at the N-terminus), 1, 2, 3, 4, 5, 8,9, 11, 12, 15, 16, 19, 22, 29, 30, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 52, 55, 57, 59, 65, 66, 69, 70, 71,74, 88, 91, 92, 94, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106,107, 108, 109, 111, 112, 113, 115, 116, 119, 120, 122, 123, 126, 127,129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,158, 159, 161, 168, 172, 183, 184, 185, 186, 187, 188, 189, 190, 191,192 (i.e. at the carboxyl terminus of the protein) or any combinationthereof from SEQ ID NO: 2.

V. Expression in Non-Eukaryotes and Eukaryotes

To obtain high level expression of a cloned hGH polynucleotide, onetypically subclones polynucleotides encoding a hGH polypeptide of theinvention into an expression vector that contains a strong promoter todirect transcription, a transcription/translation terminator, and if fora nucleic acid encoding a protein, a ribosome binding site fortranslational initiation. Suitable bacterial promoters are known tothose of ordinary skill in the art and described, e.g., in Sambrook etal. Molecular Cloning, A Laboratory Manual (2001) and Ausubel et al.Current Protocols in Molecular Biology (1999).

Bacterial expression systems for expressing hGH polypeptides of theinvention are available in, including but not limited to, E. coli,Bacillus sp., Pseudomonas fluorescens, Pseudomonas aeruginosa,Pseudomonas putida, and Salmonella (Palva et al., Gene 22:229-235(1983); Mosbach et al., Nature 302:543-545 (1983)). Kits for suchexpression systems are commercially available. Eukaryotic expressionsystems for mammalian cells, yeast, and insect cells are known to thoseof ordinary skill in the art and are also commercially available. Incases where orthogonal tRNAs and aminoacyl tRNA synthetases (describedabove) are used to express the hGH polypeptides of the invention, hostcells for expression are selected based on their ability to use theorthogonal components. Exemplary host cells include Gram-positivebacteria (including but not limited to B. brevis, B. subtilis, orStreptomyces) and Gram-negative bacteria (E. coli, Pseudomonasfluorescens, Pseudomonas aeruginosa, Pseudomonas putida), as well asyeast and other eukaryotic cells. Cells comprising O-tRNA/O-RS pairs canbe used as described herein.

A eukaryotic host cell or non-eukaryotic host cell of the presentinvention provides the ability to synthesize proteins that compriseunnatural amino acids in large useful quantities. In one aspect, thecomposition optionally includes, including but not limited to, at least10 micrograms, at least 50 micrograms, at least 75 micrograms, at least100 micrograms, at least 200 micrograms, at least 250 micrograms, atleast 500 micrograms, at least 1 milligram, at least 10 milligrams, atleast 100 milligrams, at least one gram, or more of the protein thatcomprises an unnatural amino acid, or an amount that can be achievedwith in vivo protein production methods (details on recombinant proteinproduction and purification are provided herein). In another aspect, theprotein is optionally present in the composition at a concentration of,including but not limited to, at least 10 micrograms of protein perliter, at least 50 micrograms of protein per liter, at least 75micrograms of protein per liter, at least 100 micrograms of protein perliter, at least 200 micrograms of protein per liter, at least 250micrograms of protein per liter, at least 500 micrograms of protein perliter, at least 1 milligram of protein per liter, or at least 10milligrams of protein per liter or more, in, including but not limitedto, a cell lysate, a buffer, a pharmaceutical buffer, or other liquidsuspension (including but not limited to, in a volume of, including butnot limited to, anywhere from about 1 nl to about 100 L or more). Theproduction of large quantities (including but not limited to, greaterthat that typically possible with other methods, including but notlimited to, in vitro translation) of a protein in a eukaryotic cellincluding at least one unnatural amino acid is a feature of theinvention.

A eukaryotic host cell or non-eukaryotic host cell of the presentinvention provides the ability to biosynthesize proteins that compriseunnatural amino acids in large useful quantities. For example, proteinscomprising an unnatural amino acid can be produced at a concentrationof, including but not limited to, at least 10 μg/liter, at least 50μg/liter, at least 75 μg/liter, at least 100 μg/liter, at least 200μg/liter, at least 250 μg/liter, or at least 500 μg/liter, at least 1mg/liter, at least 2 mg/liter, at least 3 mg/liter, at least 4 mg/liter,at least 5 mg/liter, at least 6 mg/liter, at least 7 mg/liter, at least8 mg/liter, at least 9 mg/liter, at least 10 mg/liter, at least 20, 30,40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900mg/liter, 1 g/liter, 5 g/liter, 10 g/liter or more of protein in a cellextract, cell lysate, culture medium, a buffer, and/or the like.

Expression systems, vectors, host cells, culturing conditions andmedium, and isolation from host cells of hGH polypeptides are furtherdescribed in U.S. patent application Ser. No. 11/046,432 entitled“Modified Human Growth Hormone Polypeptides and Their Uses,” which isincorporated by reference herein.

V. General Purification Methods

The hGH polypeptides of the present invention are normally purifiedafter expression in recombinant systems. The hGH polypeptide may bepurified from host cells by a variety of methods known to the art. hGHpolypeptides produced in bacterial host cells may be poorly soluble orinsoluble (in the form of inclusion bodies). Amino acid substitutionsmay readily be made in the hGH polypeptide that are selected for thepurpose of increasing the solubility of the recombinantly producedprotein utilizing the methods disclosed herein as well as those known inthe art. In the case of insoluble protein, the protein may be collectedfrom host cell lysates by centrifugation and may further be followed byhomogenization of the cells. In the case of poorly soluble protein,compounds including, but not limited to, polyethylene imine (PEI) may beadded to induce the precipitation of partially soluble protein. Theprecipitated protein may then be conveniently collected bycentrifugation. Recombinant host cells may be disrupted or homogenizedto release the inclusion bodies from within the cells using a variety ofmethods known to those of ordinary skill in the art. Host celldisruption or homogenization may be performed using techniques known tothose of ordinary skill in the art, including, but not limited to,enzymatic cell disruption, sonication, dounce homogenization, or highpressure release disruption. In one embodiment of the method of thepresent invention, the high pressure release technique is used todisrupt the E. coli host cells to release the inclusion bodies of thehGH polypeptides. When handling inclusion bodies of hGH polypeptide, itis advantageous to minimize the homogenization time on repetitions inorder to maximize the yield of inclusion bodies without loss due tofactors such as solubilization, mechanical shearing or proteolysis.

Insoluble or precipitated hGH polypeptide may then be solubilized usingany of a number of suitable solubilization agents known to the art. ThehGH polypeptide may be solubilized with urea or guanidine hydrochloride.The volume of the solubilized hGH polypeptide should be minimized sothat large batches may be produced using conveniently manageable batchsizes. This factor may be significant in a large-scale commercialsetting where the recombinant host may be grown in batches that arethousands of liters in volume. In addition, when manufacturing hGHpolypeptide in a large-scale commercial setting, in particular for humanpharmaceutical uses, the avoidance of harsh chemicals that can damagethe machinery and container, or the protein product itself, should beavoided, if possible. It has been shown in the method of the presentinvention that the milder denaturing agent urea can be used tosolubilize the hGH polypeptide inclusion bodies in place of the harsherdenaturing agent guanidine hydrochloride. The use of urea significantlyreduces the risk of damage to stainless steel equipment utilized in themanufacturing and purification process of hGH polypeptide whileefficiently solubilizing the hGH polypeptide inclusion bodies.

In the case of soluble hGH protein, the hGH may be secreted into theperiplasmic space or into the culture medium. In addition, soluble hGHmay be present in the cytoplasm of the host cells. It may be desired toconcentrate soluble hGH prior to performing purification steps. Standardtechniques known to those of ordinary skill in the art may be used toconcentrate soluble hGH from, for example, cell lysates or culturemedium. In addition, standard techniques known to those of ordinaryskill in the art may be used to disrupt host cells and release solublehGH from the cytoplasm or periplasmic space of the host cells.

When hGH polypeptide is produced as a fusion protein, the fusionsequence may be removed. Removal of a fusion sequence may beaccomplished by enzymatic or chemical cleavage. Enzymatic removal offusion sequences may be accomplished using methods known to those ofordinary skill in the art. The choice of enzyme for removal of thefusion sequence will be determined by the identity of the fusion, andthe reaction conditions will be specified by the choice of enzyme aswill be apparent to one of ordinary skill in the art. Chemical cleavagemay be accomplished using reagents known to those of ordinary skill inthe art, including but not limited to, cyanogen bromide, TEV protease,and other reagents. The cleaved hGH polypeptide may be purified from thecleaved fusion sequence by methods known to those of ordinary skill inthe art. Such methods will be determined by the identity and propertiesof the fusion sequence and the hGH polypeptide, as will be apparent toone of ordinary skill in the art. Methods for purification may include,but are not limited to, size-exclusion chromatography, hydrophobicinteraction chromatography, ion-exchange chromatography or dialysis orany combination thereof.

The hGH polypeptide may also be purified to remove DNA from the proteinsolution. DNA may be removed by any suitable method known to the art,such as precipitation or ion exchange chromatography, but may be removedby precipitation with a nucleic acid precipitating agent, such as, butnot limited to, protamine sulfate. The hGH polypeptide may be separatedfrom the precipitated DNA using standard methods known to those ofordinary skill in the art, including, but not limited to, centrifugationor filtration. Removal of host nucleic acid molecules is an importantfactor in a setting where the hGH polypeptide is to be used to treathumans and the methods of the present invention reduce host cell DNA topharmaceutically acceptable levels.

Methods for small-scale or large-scale fermentation can also be used inprotein expression, including but not limited to, fermentors, shakeflasks, fluidized bed bioreactors, hollow fiber bioreactors, rollerbottle culture systems, and stirred tank bioreactor systems. Each ofthese methods can be performed in a batch, fed-batch, or continuous modeprocess.

Human hGH polypeptides of the invention can generally be recovered usingmethods standard in the art. For example, culture medium or cell lysatecan be centrifuged or filtered to remove cellular debris. Thesupernatant may be concentrated or diluted to a desired volume ordiafiltered into a suitable buffer to condition the preparation forfurther purification. Further purification of the hGH polypeptide of thepresent invention includes separating deamidated and clipped forms ofthe hGH polypeptide variant from the intact form.

Any of the following exemplary procedures can be employed forpurification of hGH polypeptides of the invention: affinitychromatography; anion- or cation-exchange chromatography (using,including but not limited to, DEAE SEPHAROSE); chromatography on silica;high performance liquid chromatography (HPLC), reverse phase HPLC(RP-HPLC); gel filtration chromatography (using, including but notlimited to, SEPHADEX G-75); hydrophobic interaction chromatography;size-exclusion chromatography, metal-chelate chromatography;ultrafiltration/diafiltration; ethanol precipitation; ammonium sulfateprecipitation; chromatofocusing; displacement chromatography;electrophoretic procedures (including but not limited to preparativeisoelectric focusing), differential solubility (including but notlimited to ammonium sulfate precipitation), SDS-PAGE, or extraction.

Proteins of the present invention, including but not limited to,proteins comprising unnatural amino acids, antibodies to proteinscomprising unnatural amino acids, binding partners for proteinscomprising unnatural amino acids, etc., can be purified, eitherpartially or substantially to homogeneity, according to standardprocedures known to and used by those of skill in the art. Accordingly,polypeptides of the invention can be recovered and purified by any of anumber of methods known to those of ordinary skill in the art, includingbut not limited to, ammonium sulfate or ethanol precipitation, acid orbase extraction, column chromatography, affinity column chromatography,anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, hydroxylapatitechromatography, lectin chromatography, gel electrophoresis and the like.Protein refolding steps can be used, as desired, in making correctlyfolded mature proteins. High performance liquid chromatography (HPLC),affinity chromatography or other suitable methods can be employed infinal purification steps where high purity is desired. In oneembodiment, antibodies made against unnatural amino acids (or proteinscomprising unnatural amino acids) are used as purification reagents,including but not limited to, for affinity-based purification ofproteins comprising one or more unnatural amino acid(s). Once purified,partially or to homogeneity, as desired, the polypeptides are optionallyused for a wide variety of utilities, including but not limited to, asassay components, therapeutics, prophylaxis, diagnostics, researchreagents, and/or as immunogens for antibody production.

In addition to other references noted herein, a variety ofpurification/protein folding methods are known to those of ordinaryskill in the art, including, but not limited to, those set forth in R.Scopes, Protein Purification, Springer-Verlag, N.Y. (1982); Deutscher,Methods in Enzymology Vol. 182: Guide to Protein Purification, AcademicPress, Inc. N.Y. (1990); Sandana, (1997) Bioseparation of Proteins,Academic Press, Inc.; Bollag et al. (1996) Protein Methods, 2nd EditionWiley-Liss, NY; Walker, (1996) The Protein Protocols Handbook HumanaPress, NJ, Harris and Angal, (1990) Protein Purification Applications: APractical Approach IRL Press at Oxford, Oxford, England; Harris andAngal, Protein Purification Methods: A Practical Approach IRL Press atOxford, Oxford, England; Scopes, (1993) Protein Purification: Principlesand Practice 3rd Edition Springer Verlag, NY; Janson and Ryden, (1998)Protein Purification: Principles, High Resolution Methods andApplications, Second Edition Wiley-VCH, NY; and Walker (1998), ProteinProtocols on CD-ROM Humana Press, NJ; and the references cited therein.

One advantage of producing a protein or polypeptide of interest with anunnatural amino acid in a eukaryotic host cell or non-eukaryotic hostcell is that typically the proteins or polypeptides will be folded intheir native conformations. However, in certain embodiments of theinvention, those of skill in the art will recognize that, aftersynthesis, expression and/or purification, proteins can possess aconformation different from the desired conformations of the relevantpolypeptides. In one aspect of the invention, the expressed protein isoptionally denatured and then renatured. This is accomplished utilizingmethods known in the art, including but not limited to, by adding achaperonin to the protein or polypeptide of interest, by solubilizingthe proteins in a chaotropic agent such as guanidine HCl, utilizingprotein disulfide isomerase, etc.

In general, it is occasionally desirable to denature and reduceexpressed polypeptides and then to cause the polypeptides to re-foldinto the preferred conformation. For example, guanidine, urea, DTT, DTE,and/or a chaperonin can be added to a translation product of interest.Methods of reducing, denaturing and renaturing proteins are known tothose of ordinary skill in the art (see, the references above, andDebinski, et al. (1993) J. Biol. Chem., 268: 14065-14070; Kreitman andPastan (1993) Bioconjug. Chem., 4: 581-585; and Buchner, et al., (1992)Anal. Biochem., 205: 263-270). Debinski, et al., for example, describethe denaturation and reduction of inclusion body proteins inguanidine-DTE. The proteins can be refolded in a redox buffercontaining, including but not limited to, oxidized glutathione andL-arginine. Refolding reagents can be flowed or otherwise moved intocontact with the one or more polypeptide or other expression product, orvice-versa.

In the case of prokaryotic production of hGH polypeptide, the hGHpolypeptide thus produced may be misfolded and thus lacks or has reducedbiological activity. The bioactivity of the protein may be restored by“refolding”. In general, misfolded hGH polypeptide is refolded bysolubilizing (where the hGH polypeptide is also insoluble), unfoldingand reducing the polypeptide chain using, for example, one or morechaotropic agents (e.g. urea and/or guanidine) and a reducing agentcapable of reducing disulfide bonds (e.g. dithiothreitol, DTT or2-mercaptoethanol, 2-ME). At a moderate concentration of chaotrope, anoxidizing agent is then added (e.g., oxygen, cystine or cystamine),which allows the reformation of disulfide bonds. hGH polypeptide may berefolded using standard methods known in the art, such as thosedescribed in U.S. Pat. Nos. 4,511,502, 4,511,503, and 4,512,922, whichare incorporated by reference herein. The hGH polypeptide may also becofolded with other proteins to form heterodimers or heteromultimers.

After refolding or cofolding, the hGH polypeptide may be furtherpurified. Purification of hGH may be accomplished using a variety oftechniques known to those of ordinary skill in the art, includinghydrophobic interaction chromatography, size exclusion chromatography,ion exchange chromatography, reverse-phase high performance liquidchromatography, affinity chromatography, and the like or any combinationthereof. Additional purification may also include a step of drying orprecipitation of the purified protein.

After purification, hGH may be exchanged into different buffers and/orconcentrated by any of a variety of methods known to those of ordinaryskill in the art, including, but not limited to, diafiltration anddialysis. hGH that is provided as a single purified protein may besubject to aggregation and precipitation.

The purified hGH may be at least 90% pure (as measured by reverse phasehigh performance liquid chromatography, RP-HPLC, or sodium dodecylsulfate-polyacrylamide gel electrophoresis, SDS-PAGE) or at least 95%pure, or at least 98% pure, or at least 99% or greater pure. Regardlessof the exact numerical value of the purity of the hGH, the hGH issufficiently pure for use as a pharmaceutical product or for furtherprocessing, such as conjugation with a water soluble polymer such asPEG.

Certain hGH molecules may be used as therapeutic agents in the absenceof other active ingredients or proteins (other than excipients,carriers, and stabilizers, serum albumin and the like), or they may becomplexed with another protein or a polymer.

Any one of a variety of isolation steps may be performed on the celllysate, extract, culture medium, inclusion bodies, periplasmic space ofthe host cells, cytoplasm of the host cells, or other material,comprising hGH polypeptide or on any hGH polypeptide mixtures resultingfrom any isolation steps including, but not limited to, affinitychromatography, ion exchange chromatography, hydrophobic interactionchromatography, gel filtration chromatography, high performance liquidchromatography (“HPLC”), reversed phase-HPLC (“RP-HPLC”), expanded bedadsorption, or any combination and/or repetition thereof and in anyappropriate order.

Equipment and other necessary materials used in performing thetechniques described herein are commercially available. Pumps, fractioncollectors, monitors, recorders, and entire systems are available from,for example, Applied Biosystems (Foster City, Calif.), Bio-RadLaboratories, Inc. (Hercules, Calif.), and Amersham Biosciences, Inc.(Piscataway, N.J.). Chromatographic materials including, but not limitedto, exchange matrix materials, media, and buffers are also availablefrom such companies.

Equilibration, and other steps in the column chromatography processesdescribed herein such as washing and elution, may be more rapidlyaccomplished using specialized equipment such as a pump. Commerciallyavailable pumps include, but are not limited to, HILOAD® Pump P-50,Peristaltic Pump P-1, Pump P-901, and Pump P-903 (Amersham Biosciences,Piscataway, N.J.).

Examples of fraction collectors include RediFrac Fraction Collector,FRAC-100 and FRAC-200 Fraction Collectors, and SUPERFRAC® FractionCollector (Amersham Biosciences, Piscataway, N.J.). Mixers are alsoavailable to form pH and linear concentration gradients. Commerciallyavailable mixers include Gradient Mixer GM-1 and In-Line Mixers(Amersham Biosciences, Piscataway, N.J.).

The chromatographic process may be monitored using any commerciallyavailable monitor. Such monitors may be used to gather information likeUV, pH, and conductivity. Examples of detectors include Monitor UV-1,UVICORD® S II, Monitor UV-M II, Monitor UV-900, Monitor UPC-900, MonitorpH/C-900, and Conductivity Monitor (Amersham Biosciences, Piscataway,N.J.). Indeed, entire systems are commercially available including thevarious AKTA® systems from Amersham Biosciences (Piscataway, N.J.).

In one embodiment of the present invention, for example, the hGHpolypeptide may be reduced and denatured by first denaturing theresultant purified hGH polypeptide in urea, followed by dilution intoTRIS buffer containing a reducing agent (such as DTT) at a suitable pH.In another embodiment, the hGH polypeptide is denatured in urea in aconcentration range of between about 2 M to about 9 M, followed bydilution in TRIS buffer at a pH in the range of about 5.0 to about 8.0.The refolding mixture of this embodiment may then be incubated. In oneembodiment, the refolding mixture is incubated at room temperature forfour to twenty-four hours. The reduced and denatured hGH polypeptidemixture may then be further isolated or purified.

As stated herein, the pH of the first hGH polypeptide mixture may beadjusted prior to performing any subsequent isolation steps. Inaddition, the first hGH polypeptide mixture or any subsequent mixturethereof may be concentrated using techniques known in the art. Moreover,the elution buffer comprising the first hGH polypeptide mixture or anysubsequent mixture thereof may be exchanged for a buffer suitable forthe next isolation step using techniques known to those of ordinaryskill in the art.

Ion Exchange Chromatography Ion exchange chromatography may beperformed. See generally ION EXCHANGE CHROMATOGRAPHY: PRINCIPLES ANDMETHODS (Cat. No. 18-1114-21, Amersham Biosciences (Piscataway, N.J.)).Commercially available ion exchange columns include HITRAP®, HIPREP®,and HILOAD® Columns (Amersham Biosciences, Piscataway, N.J.). Suchcolumns utilize strong anion exchangers such as Q SEPHAROSE® Fast Flow,Q SEPHAROSE® High Performance, and Q SEPHAROSE® XL; strong cationexchangers such as SP SEPHAROSE® High Performance, SP SEPHAROSE® FastFlow, and SP SEPHAROSE® XL; weak anion exchangers such as DEAESEPHAROSE® Fast Flow; and weak cation exchangers such as CM SEPHAROSE®Fast Flow (Amersham Biosciences, Piscataway, N.J.). Anion or cationexchange column chromatography may be performed on the hGH polypeptideat any stage of the purification process to isolate substantiallypurified hGH polypeptide.

The cation exchange chromatography step may be performed using anysuitable cation exchange matrix. Useful cation exchange matricesinclude, but are not limited to, fibrous, porous, non-porous,microgranular, beaded, or cross-linked cation exchange matrix materials.Such cation exchange matrix materials include, but are not limited to,cellulose, agarose, dextran, polyacrylate, polyvinyl, polystyrene,silica, polyether, or composites of any of the foregoing.

The cation exchange matrix may be any suitable cation exchangerincluding strong and weak cation exchangers. Strong cation exchangersmay remain ionized over a wide pH range and thus, may be capable ofbinding hGH over a wide pH range. Weak cation exchangers, however, maylose ionization as a function of pH. For example, a weak cationexchanger may lose charge when the pH drops below about pH 4 or pH 5.Suitable strong cation exchangers include, but are not limited to,charged functional groups such as sulfopropyl (SP), methyl sulfonate(S), or sulfoethyl (SE). The cation exchange matrix may be a strongcation exchanger, having an hGH binding pH range of about 2.5 to about6.0. Alternatively, the strong cation exchanger may have an hGH bindingpH range of about pH 2.5 to about pH 5.5. The cation exchange matrix maybe a strong cation exchanger having an hGH binding pH of about 3.0.Alternatively, the cation exchange matrix may be a strong cationexchanger, having an hGH binding pH range of about 6.0 to about 8.0. Thecation exchange matrix may be a strong cation exchanger having an hGHbinding pH range of about 8.0 to about 12.5. Alternatively, the strongcation exchanger may have an hGH binding pH range of about pH 8.0 toabout pH 12.0.

Prior to loading the hGH, the cation exchange matrix may beequilibrated, for example, using several column volumes of a dilute,weak acid, e.g., four column volumes of 20 mM acetic acid, pH 3.Following equilibration, the hGH may be added and the column may bewashed one to several times, prior to elution of substantially purifiedhGH, also using a weak acid solution such as a weak acetic acid orphosphoric acid solution. For example, approximately 2-4 column volumesof 20 mM acetic acid, pH 3, may be used to wash the column. Additionalwashes using, e.g., 2-4 column volumes of 0.05 M sodium acetate, pH 5.5,or 0.05 M sodium acetate mixed with 0.1 M sodium chloride, pH 5.5, mayalso be used. Alternatively, using methods known in the art, the cationexchange matrix may be equilibrated using several column volumes of adilute, weak base.

Alternatively, substantially purified hGH may be eluted by contactingthe cation exchanger matrix with a buffer having a sufficiently low pHor ionic strength to displace the hGH from the matrix. The pH of theelution buffer may range from about pH 2.5 to about pH 6.0. Morespecifically, the pH of the elution buffer may range from about pH 2.5to about pH 5.5, about pH 2.5 to about pH 5.0. The elution buffer mayhave a pH of about 3.0. In addition, the quantity of elution buffer mayvary widely and will generally be in the range of about 2 to about 10column volumes. Moreover, suitable buffers known to those of skill inthe art may include, but not limited to, citrate, phosphate, formate,HEPES, and MES buffers ranging in concentration from at least about 5 mMto at least about 100 mM.

Following adsorption of the hGH to the cation exchanger matrix,substantially purified hGH may be eluted by contacting the matrix with abuffer having a sufficiently high pH or ionic strength to displace thehGH from the matrix. The pH of the elution buffer may range from aboutpH 8.0 to about pH 12.5. More specifically, the elution buffer may rangefrom about pH 8.0 to about pH 12.0. Suitable buffers for use in high pHelution of substantially purified hGH include, but are not limited to,citrate, phosphate, formate, acetate, HEPES, and MES buffers ranging inconcentration from at least about 5 mM to at least about 100 mM. Inaddition, a buffer having 0.1 M potassium borate, 0.6 M potassiumchloride, 0.1 mM EDTA, pH 8.7 may be used. Substantially purified hGHmay also be eluted using standard buffers, such as a bicine buffer whichincludes about 50 to 100 mM bicine, about 75 mM bicine; 25 to about 100mM sodium chloride, about 50 mM sodium chloride, and about 0.05 to about0.5 EDTA, about 0.1 mM EDTA, pH 7.5.

Reverse-Phase Chromatography RP-HPLC may be performed to purify proteinsfollowing suitable protocols that are known to those of ordinary skillin the art. See, e.g., Pearson et al., ANAL BIOCHEM. (1982) 124:217-230(1982); Rivier et al., J. CHROM. (1983) 268:112-119; Kunitani et al., J.CHROM. (1986) 359:391-402. RP-HPLC may be performed on the hGHpolypeptide to isolate substantially purified hGH polypeptide. In thisregard, silica derivatized resins with alkyl functionalities with a widevariety of lengths, including, but not limited to, at least about C₃ toat least about C₃₀, at least about C₃ to at least about C₂₀, or at leastabout C₃ to at least about C₁₈, resins may be used. Alternatively, apolymeric resin may be used. For example, TosoHaas Amberchrome CG 1000sdresin may be used, which is a styrene polymer resin. Cyano or polymericresins with a wide variety of alkyl chain lengths may also be used.Furthermore, the RP-HPLC column may be washed with a solvent such asethanol. The Source RP column is another example of a RP-HPLC column.

A suitable elution buffer containing an ion pairing agent and an organicmodifier such as methanol, isopropanol, tetrahydrofuran, acetonitrile orethanol, may be used to elute the hGH polypeptide from the RP-HPLCcolumn. The most commonly used ion pairing agents include, but are notlimited to, acetic acid, formic acid, perchloric acid, phosphoric acid,trifluoroacetic acid, heptafluorobutyric acid, triethylamine,tetramethylammonium, tetrabutylammonium, and triethylammonium acetate.Elution may be performed using one or more gradients or isocraticconditions, with gradient conditions preferred to reduce the separationtime and to decrease peak width. Another method involves the use of twogradients with different solvent concentration ranges. Examples ofsuitable elution buffers for use herein may include, but are not limitedto, ammonium acetate and acetonitrile solutions.

hGH may be isolated or purified, for example, using a SOURCE RP column,with an acetonitrile gradient.

Hydrophobic Interaction Chromatography Purification TechniquesHydrophobic interaction chromatography (HIC) may be performed on the hGHpolypeptide. See generally HYDROPHOBIC INTERACTION CHROMATOGRAPHYHANDBOOK: PRINCIPLES AND METHODS (Cat. No. 18-1020-90, AmershamBiosciences (Piscataway, N.J.) which is incorporated by referenceherein. Suitable HIC matrices may include, but are not limited to,alkyl- or aryl-substituted matrices, such as butyl-, hexyl-, octyl- orphenyl-substituted matrices including agarose, cross-linked agarose,sepharose, cellulose, silica, dextran, polystyrene, poly(methacrylate)matrices, and mixed mode resins, including but not limited to, apolyethyleneamine resin or a butyl- or phenyl-substitutedpoly(methacrylate) matrix. Commercially available sources forhydrophobic interaction column chromatography include, but are notlimited to, HITRAP®, HIPREP®, and HILOAD® columns (Amersham Biosciences,Piscataway, N.J.).

Briefly, prior to loading, the HIC column may be equilibrated usingstandard buffers known to those of ordinary skill in the art, such as anacetic acid/sodium chloride solution or HEPES containing ammoniumsulfate. Ammonium sulfate may be used as the buffer for loading the HICcolumn. After loading the hGH polypeptide, the column may then washedusing standard buffers and conditions to remove unwanted materials butretaining the hGH polypeptide on the HIC column. The hGH polypeptide maybe eluted with about 3 to about 10 column volumes of a standard buffer,such as a HEPES buffer containing EDTA and lower ammonium sulfateconcentration than the equilibrating buffer, or an acetic acid/sodiumchloride buffer, among others. A decreasing linear salt gradient using,for example, a gradient of potassium phosphate, may also be used toelute the hGH molecules. The eluant may then be concentrated, forexample, by filtration such as diafiltration or ultrafiltration.Diafiltration may be utilized to remove the salt used to elute the hGHpolypeptide.

Isolation steps using, for example, gel filtration (GEL FILTRATION:PRINCIPLES AND METHODS (Cat. No. 18-1022-18, Amersham Biosciences,Piscataway, N.J.) which is incorporated by reference herein,hydroxyapatite chromatography (suitable matrices include, but are notlimited to, HA-Ultrogel, High Resolution (Calbiochem), CHT CeramicHydroxyapatite (BioRad), Bio-Gel HTP Hydroxyapatite (BioRad)), HPLC,expanded bed adsorption, ultrafiltration, diafiltration, lyophilization,and the like, may be performed on the first hGH polypeptide mixture orany subsequent mixture thereof, to remove any excess salts and toreplace the buffer with a suitable buffer for the next isolation step oreven formulation of the final drug product.

The yield of hGH polypeptide, including substantially purified hGHpolypeptide, may be monitored at each step described herein usingtechniques known to those of ordinary skill in the art. Such techniquesmay also be used to assess the yield of substantially purified hGHpolypeptide following the last isolation step. For example, the yield ofhGH polypeptide may be monitored using any of several reverse phase highpressure liquid chromatography columns, having a variety of alkyl chainlengths such as cyano RP-HPLC, C₁₈RP-HPLC; as well as cation exchangeHPLC and gel filtration HPLC.

Purity may be determined using standard techniques, such as SDS-PAGE, orby measuring hGH polypeptide using Western blot and ELISA assays. Forexample, polyclonal antibodies may be generated against proteinsisolated from negative control yeast fermentation and the cationexchange recovery. The antibodies may also be used to probe for thepresence of contaminating host cell proteins.

RP-HPLC material Vydac C4 (Vydac) consists of silica gel particles, thesurfaces of which carry C4-alkyl chains. The separation of hGHpolypeptide from the proteinaceous impurities is based on differences inthe strength of hydrophobic interactions. Elution is performed with anacetonitrile gradient in diluted trifluoroacetic acid. Preparative HPLCis performed using a stainless steel column (filled with 2.8 to 3.2liter of Vydac C4 silicagel). The Hydroxyapatite Ultrogel eluate isacidified by adding trifluoroacetic acid and loaded onto the Vydac C4column. For washing and elution an acetonitrile gradient in dilutedtrifluoroacetic acid is used. Fractions are collected and immediatelyneutralized with phosphate buffer. The hGH polypeptide fractions whichare within the IPC limits are pooled.

DEAE Sepharose (GE Healthcare) material consists of diethylaminoethyl(DEAE)-groups which are covalently bound to the surface of Sepharosebeads. The binding of hGH polypeptide to the DEAE groups is mediated byionic interactions. Acetonitrile and trifluoroacetic acid pass throughthe column without being retained. After these substances have beenwashed off, trace impurities are removed by washing the column withacetate buffer at a low pH. Then the column is washed with neutralphosphate buffer and hGH polypeptide is eluted with a buffer withincreased ionic strength. The column is packed with DEAE Sepharose fastflow. The column volume is adjusted to assure a hGH polypeptide load inthe range of 3-10 mg hGH polypeptide/ml gel. The column is washed withwater and equilibration buffer (sodium/potassium phosphate). The pooledfractions of the HPLC eluate are loaded and the column is washed withequilibration buffer. Then the column is washed with washing buffer(sodium acetate buffer) followed by washing with equilibration buffer.Subsequently, hGH polypeptide is eluted from the column with elutionbuffer (sodium chloride, sodium/potassium phosphate) and collected in asingle fraction in accordance with the master elution profile. Theeluate of the DEAE Sepharose column is adjusted to the specifiedconductivity. The resulting drug substance is sterile filtered intoTeflon bottles and stored at −70° C.

Additional methods that may be employed include, but are not limited to,steps to remove endotoxins. Endotoxins are lipopoly-saccharides (LPSs)which are located on the outer membrane of Gram-negative host cells,such as, for example, Escherichia coli. Methods for reducing endotoxinlevels are known to one of ordinary skill in the art and include, butare not limited to, purification techniques using silica supports, glasspowder or hydroxyapatite, reverse-phase, affinity, size-exclusion,anion-exchange chromatography, hydrophobic interaction chromatography, acombination of these methods, and the like. Modifications or additionalmethods may be required to remove contaminants such as co-migratingproteins from the polypeptide of interest. Methods for measuringendotoxin levels are known to one of ordinary skill in the art andinclude, but are not limited to, Limulus Amebocyte Lysate (LAL) assays.

A wide variety of methods and procedures can be used to assess the yieldand purity of a hGH protein one or more non-naturally encoded aminoacids, including but not limited to, the Bradford assay, SDS-PAGE,silver stained SDS-PAGE, coomassie stained SDS-PAGE, mass spectrometry(including but not limited to, MALDI-TOF) and other methods forcharacterizing proteins known to one of ordinary skill in the art.Additional methods include, but are not limited to: SDS-PAGE coupledwith protein staining methods, immunoblotting, matrix assisted laserdesorption/ionization-mass spectrometry (MALDI-MS), liquidchromatography/mass spectrometry, isoelectric focusing, analytical anionexchange, chromatofocusing, and circular dichroism.

Procedures including, but not limited to, those listed herein forisolation and purification may also be used in formulation studies toassess the stability of hGH polypeptides of the invention, the stabilityof PEGylated forms of hGH polypeptides, and progress of the PEGylationreaction.

VII. Expression in Alternate Systems

Several strategies have been employed to introduce unnatural amino acidsinto proteins in non-recombinant host cells, mutagenized host cells, orin cell-free systems. These systems are also suitable for use in makingthe hGH polypeptides comprising a non-naturally encoded amino acid.Derivatization of amino acids with reactive side-chains such as Lys, Cysand Tyr resulted in the conversion of lysine to N²-acetyl-lysine.Chemical synthesis also provides a straightforward method to incorporateunnatural amino acids. With the recent development of enzymatic ligationand native chemical ligation of peptide fragments, it is possible tomake larger proteins. See, e.g., P. E. Dawson and S. B. H. Kent, Annu.Rev. Biochem, 69:923 (2000). Chemical peptide ligation and nativechemical ligation are described in U.S. Pat. No. 6,184,344, U.S. PatentPublication No. 2004/0138412, U.S. Patent Publication No. 2003/0208046,WO 02/098902, and WO 03/042235, which are incorporated by referenceherein. A general in vitro biosynthetic method in which a suppressortRNA chemically acylated with the desired unnatural amino acid is addedto an in vitro extract capable of supporting protein biosynthesis, hasbeen used to site-specifically incorporate over 100 unnatural aminoacids into a variety of proteins of virtually any size. See, e.g., V. W.Cornish, D. Mendel and P. G. Schultz, Angew. Chem. Int. Ed. Engl., 1995,34:621 (1995); C. J. Noren, S. J. Anthony-Cahill, M. C. Griffith, P. G.Schultz, A general method for site-specific incorporation of unnaturalamino acids into proteins, Science 244:182-188 (1989); and, J. D. Bain,C. G. Glabe, T. A. Dix, A. R. Chamberlin, E. S. Diala, Biosyntheticsite-specific incorporation of a non-natural amino acid into apolypeptide, J. Am. Chem. Soc. 111:8013-8014 (1989). A broad range offunctional groups has been introduced into proteins for studies ofprotein stability, protein folding, enzyme mechanism, and signaltransduction.

An in vivo method, termed selective pressure incorporation, wasdeveloped to exploit the promiscuity of wild-type synthetases. See,e.g., N. Budisa, C. Minks, S. Alefelder, W. Wenger, F. M. Dong, L.Moroder and R. Huber, FASEB J., 13:41 (1999). An auxotrophic strain, inwhich the relevant metabolic pathway supplying the cell with aparticular natural amino acid is switched off, is grown in minimal mediacontaining limited concentrations of the natural amino acid, whiletranscription of the target gene is repressed. At the onset of astationary growth phase, the natural amino acid is depleted and replacedwith the unnatural amino acid analog. Induction of expression of therecombinant protein results in the accumulation of a protein containingthe unnatural analog. For example, using this strategy, o, m andp-fluorophenylalanines have been incorporated into proteins, and exhibittwo characteristic shoulders in the UV spectrum which can be easilyidentified, see, e.g., C. Minks, R. Huber, L. Moroder and N. Budisa,Anal. Biochem., 284:29 (2000); trifluoromethionine has been used toreplace methionine in bacteriophage T4 lysozyme to study its interactionwith chitooligosaccharide ligands by ¹⁹F NMR, see, e.g., H. Duewel, E.Daub, V. Robinson and J. F. Honek, Biochemistry, 36:3404 (1997); andtrifluoroleucine has been incorporated in place of leucine, resulting inincreased thermal and chemical stability of a leucine-zipper protein.See, e.g., Y. Tang, G. Ghirlanda, W. A. Petka, T. Nakajima, W. F.DeGrado and D. A. Tirrell, Angew. Chem. Int. Ed. Engl., 40:1494 (2001).Moreover, selenomethionine and telluromethionine are incorporated intovarious recombinant proteins to facilitate the solution of phases inX-ray crystallography. See, e.g., W. A. Hendrickson, J. R. Horton and D.M. Lemaster, EMBO J., 9:1665 (1990); J. O. Boles, K. Lewinski, M.Kunkle, J. D. Odom, B. Dunlap, L. Lebioda and M. Hatada, Nat. Struct.Biol., 1:283 (1994); N. Budisa, B. Steipe, P. Demange, C. Eckerskorn, J.Kellermann and R. Huber, Eur. J. Biochem., 230:788 (1995); and, N.Budisa, W. Karnbrock, S. Steinbacher, A. Humm, L. Prade, T. Neuefeind,L. Moroder and R. Huber, J. Mol. Biol., 270:616 (1997). Methionineanalogs with alkene or alkyne functionalities have also beenincorporated efficiently, allowing for additional modification ofproteins by chemical means. See, e.g., J. C. van Hest and D. A. Tirrell,FEBS Lett., 428:68 (1998); J. C. van Hest, K. L. Kiick and D. A.Tirrell, J. Am. Chem. Soc., 122:1282 (2000); and, K. L. Kiick and D. A.Tirrell, Tetrahedron, 56:9487 (2000); U.S. Pat. No. 6,586,207; U.S.Patent Publication 2002/0042097, which are incorporated by referenceherein.

The success of this method depends on the recognition of the unnaturalamino acid analogs by aminoacyl-tRNA synthetases, which, in general,require high selectivity to insure the fidelity of protein translation.One way to expand the scope of this method is to relax the substratespecificity of aminoacyl-tRNA synthetases, which has been achieved in alimited number of cases. For example, replacement of Ala²⁹⁴ by Gly inEscherichia coli phenylalanyl-tRNA synthetase (PheRS) increases the sizeof substrate binding pocket, and results in the acylation of tRNAPhe byp-Cl-phenylalanine (p-Cl-Phe). See, M. Ibba, P. Kast and H. Hennecke,Biochemistry, 33:7107 (1994). An Escherichia coli strain harboring thismutant PheRS allows the incorporation of p-Cl-phenylalanine orp-Br-phenylalanine in place of phenylalanine. See, e.g., M. Ibba and H.Hennecke, FEBS Lett., 364:272 (1995); and, N. Sharma, R. Furter, P. Kastand D. A. Tirrell, FEBS Lett., 467:37 (2000). Similarly, a pointmutation Phe130Ser near the amino acid binding site of Escherichia colityrosyl-tRNA synthetase was shown to allow azatyrosine to beincorporated more efficiently than tyrosine. See, F. Hamano-Takaku, T.Iwama, S. Saito-Yano, K. Takaku, Y. Monden, M. Kitabatake, D. Soll andS. Nishimura, J. Biol. Chem., 275:40324 (2000).

Another strategy to incorporate unnatural amino acids into proteins invivo is to modify synthetases that have proofreading mechanisms. Thesesynthetases cannot discriminate and therefore activate amino acids thatare structurally similar to the cognate natural amino acids. This erroris corrected at a separate site, which deacylates the mischarged aminoacid from the tRNA to maintain the fidelity of protein translation. Ifthe proofreading activity of the synthetase is disabled, structuralanalogs that are misactivated may escape the editing function and beincorporated. This approach has been demonstrated recently with thevalyl-tRNA synthetase (ValRS). See, V. Doring, H. D. Mootz, L. A.Nangle, T. L. Hendrickson, V. de Crecy-Lagard, P. Schimmel and P.Marliere, Science, 292:501 (2001). ValRS can misaminoacylate tRNAValwith Cys, Thr, or aminobutyrate (Abu); these noncognate amino acids aresubsequently hydrolyzed by the editing domain. After random mutagenesisof the Escherichia coli chromosome, a mutant Escherichia coli strain wasselected that has a mutation in the editing site of ValRS. Thisedit-defective ValRS incorrectly charges tRNAVal with Cys. Because Abusterically resembles Cys (—SH group of Cys is replaced with —CH3 inAbu), the mutant ValRS also incorporates Abu into proteins when thismutant Escherichia coli strain is grown in the presence of Abu. Massspectrometric analysis shows that about 24% of valines are replaced byAbu at each valine position in the native protein.

Solid-phase synthesis and semisynthetic methods have also allowed forthe synthesis of a number of proteins containing novel amino acids. Forexample, see the following publications and references cited within,which are as follows: Crick, F. H. C., Barrett, L. Brenner, S.Watts-Tobin, R. General nature of the genetic code for proteins. Nature,192:1227-1232 (1961); Hofmann, K., Bohn, H. Studies on polypeptides.XXXVI. The effect of pyrazole-imidazole replacements on the S-proteinactivating potency of an S-peptide fragment, J. Am. Chem,88(24):5914-5919 (1966); Kaiser, E. T. Synthetic approaches tobiologically active peptides and proteins including enyzmes, Acc ChemRes, 22:47-54 (1989); Nakatsuka, T., Sasaki, T., Kaiser, E. T. Peptidesegment coupling catalyzed by the semisynthetic enzyme thiosubtilisin, JAm Chem Soc, 109:3808-3810 (1987); Schnolzer, M., Kent, S B H.Constructing proteins by dovetailing unprotected synthetic peptides:backbone-engineered HIV protease, Science, 256(5054):221-225 (1992);Chaiken, I. M. Semisynthetic peptides and proteins, CRC Crit RevBiochem, 11(3):255-301 (1981); Offord, R. E. Protein engineering bychemical means? Protein Eng., 1(3):151-157 (1987); and, Jackson, D. Y.,Burnier, J., Quan, C., Stanley, M., Tom, J., Wells, J. A. A DesignedPeptide Ligase for Total Synthesis of Ribonuclease A with UnnaturalCatalytic Residues, Science, 266(5183):243 (1994).

Chemical modification has been used to introduce a variety of unnaturalside chains, including cofactors, spin labels and oligonucleotides intoproteins in vitro. See, e.g., Corey, D. R., Schultz, P. G. Generation ofa hybrid sequence-specific single-stranded deoxyribonuclease, Science,238(4832):1401-1403 (1987); Kaiser, E. T., Lawrence D. S., Rokita, S. E.The chemical modification of enzymatic specificity, Annu Rev Biochem,54:565-595 (1985); Kaiser, E. T., Lawrence, D. S. Chemical mutation ofenzyme active sites, Science, 226(4674):505-511 (1984); Neet, K. E.,Nanci A, Koshland, D. E. Properties of thiol-subtilisin, J. Biol. Chem.,243(24):6392-6401 (1968); Polgar, L. et M. L. Bender. A new enzymecontaining a synthetically formed active site. Thiol-subtilisin. J. Am.Chem Soc, 88:3153-3154 (1966); and, Pollack, S. J., Nakayama, G.Schultz, P. G. Introduction of nucleophiles and spectroscopic probesinto antibody combining sites, Science, 242(4881):1038-1040 (1988).

Alternatively, biosynthetic methods that employ chemically modifiedaminoacyl-tRNAs have been used to incorporate several biophysical probesinto proteins synthesized in vitro. See the following publications andreferences cited within: Brunner, J. New Photolabeling and crosslinkingmethods, Annu. Rev Biochem, 62:483-514 (1993); and, Krieg, U. C.,Walter, P., Hohnson, A. E. Photocrosslinking of the signal sequence ofnascent preprolactin of the 54-kilodalton polypeptide of the signalrecognition particle, Proc. Natl. Acad. Sci, 83(22):8604-8608 (1986).

Previously, it has been shown that unnatural amino acids can besite-specifically incorporated into proteins in vitro by the addition ofchemically aminoacylated suppressor tRNAs to protein synthesis reactionsprogrammed with a gene containing a desired amber nonsense mutation.Using these approaches, one can substitute a number of the common twentyamino acids with close structural homologues, e.g., fluorophenylalaninefor phenylalanine, using strains auxotropic for a particular amino acid.See, e.g., Noren, C. J., Anthony-Cahill, Griffith, M. C., Schultz, P. G.A general method for site-specific incorporation of unnatural aminoacids into proteins, Science, 244: 182-188 (1989); M. W. Nowak, et al.,Science 268:439-42 (1995); Bain, J. D., Glabe, C. G., Dix, T. A.,Chamberlin, A. R., Diala, E. S. Biosynthetic site-specific Incorporationof a non-natural amino acid into a polypeptide, J. Am. Chem Soc,111:8013-8014 (1989); N. Budisa et al., FASEB J. 13:41-51 (1999);Ellman, J. A., Mendel, D., Anthony-Cahill, S., Noren, C. J., Schultz, P.G. Biosynthetic method for introducing unnatural amino acidssite-specifically into proteins, Methods in Enz., vol. 202, 301-336(1992); and, Mendel, D., Cornish, V. W. & Schultz, P. G. Site-DirectedMutagenesis with an Expanded Genetic Code, Annu Rev Biophys. BiomolStruct. 24, 435-62 (1995).

For example, a suppressor tRNA was prepared that recognized the stopcodon UAG and was chemically aminoacylated with an unnatural amino acid.Conventional site-directed mutagenesis was used to introduce the stopcodon TAG, at the site of interest in the protein gene. See, e.g.,Sayers, J. R., Schmidt, W. Eckstein, F. 5′-3′ Exonucleases inphosphorothioate-based olignoucleotide-directed mutagensis, NucleicAcids Res, 16(3):791-802 (1988). When the acylated suppressor tRNA andthe mutant gene were combined in an in vitro transcription/translationsystem, the unnatural amino acid was incorporated in response to the UAGcodon which gave a protein containing that amino acid at the specifiedposition. Experiments using [³H]-Phe and experiments with α-hydroxyacids demonstrated that only the desired amino acid is incorporated atthe position specified by the UAG codon and that this amino acid is notincorporated at any other site in the protein. See, e.g., Noren, et al,supra; Kobayashi et al., (2003) Nature Structural Biology 10(6):425-432;and, Ellman, J. A., Mendel, D., Schultz, P. G. Site-specificincorporation of novel backbone structures into proteins, Science,255(5041): 197-200 (1992).

A tRNA may be aminoacylated with a desired amino acid by any method ortechnique, including but not limited to, chemical or enzymaticaminoacylation.

Aminoacylation may be accomplished by aminoacyl tRNA synthetases or byother enzymatic molecules, including but not limited to, ribozymes. Theterm “ribozyme” is interchangeable with “catalytic RNA.” Cech andcoworkers (Cech, 1987, Science, 236:1532-1539; McCorkle et al., 1987,Concepts Biochem. 64:221-226) demonstrated the presence of naturallyoccurring RNAs that can act as catalysts (ribozymes). However, althoughthese natural RNA catalysts have only been shown to act on ribonucleicacid substrates for cleavage and splicing, the recent development ofartificial evolution of ribozymes has expanded the repertoire ofcatalysis to various chemical reactions. Studies have identified RNAmolecules that can catalyze aminoacyl-RNA bonds on their own(2′)3′-termini (Illangakekare et al., 1995 Science 267:643-647), and anRNA molecule which can transfer an amino acid from one RNA molecule toanother (Lohse et al., 1996, Nature 381:442-444).

U.S. Patent Application Publication 2003/0228593, which is incorporatedby reference herein, describes methods to construct ribozymes and theiruse in aminoacylation of tRNAs with naturally encoded and non-naturallyencoded amino acids. Substrate-immobilized forms of enzymatic moleculesthat can aminoacylate tRNAs, including but not limited to, ribozymes,may enable efficient affinity purification of the aminoacylatedproducts. Examples of suitable substrates include agarose, sepharose,and magnetic beads. The production and use of a substrate-immobilizedform of ribozyme for aminoacylation is described in Chemistry andBiology 2003, 10:1077-1084 and U.S. Patent Application Publication2003/0228593, which are incorporated by reference herein.

Chemical aminoacylation methods include, but are not limited to, thoseintroduced by Hecht and coworkers (Hecht, S. M. Acc. Chem. Res. 1992,25, 545; Heckler, T. G.; Roesser, J. R.; Xu, C.; Chang, P.; Hecht, S. M.Biochemistry 1988, 27, 7254; Hecht, S. M.; Alford, B. L.; Kuroda, Y.;Kitano, S. J. Biol. Chem. 1978, 253, 4517) and by Schultz, Chamberlin,Dougherty and others (Cornish, V. W.; Mendel, D.; Schultz, P. G. Angew.Chem. Int. Ed. Engl. 1995, 34, 621; Robertson, S. A.; Ellman, J. A.;Schultz, P. G. J. Am. Chem. Soc. 1991, 113, 2722; Noren, C. J.;Anthony-Cahill, S. J.; Griffith, M. C.; Schultz, P. G. Science 1989,244, 182; Bain, J. D.; Glabe, C. G.; Dix, T. A.; Chamberlin, A. R. J.Am. Chem. Soc. 1989, 111, 8013; Bain, J. D. et al. Nature 1992, 356,537; Gallivan, J. P.; Lester, H. A.; Dougherty, D. A. Chem. Biol. 1997,4, 740; Turcatti, et al. J. Biol. Chem. 1996, 271, 19991; Nowak, M. W.et al. Science, 1995, 268, 439; Saks, M. E. et al. J. Biol. Chem. 1996,271, 23169; Hohsaka, T. et al. J. Am. Chem. Soc. 1999, 121, 34), whichare incorporated by reference herein, to avoid the use of synthetases inaminoacylation. Such methods or other chemical aminoacylation methodsmay be used to aminoacylate tRNA molecules.

Methods for generating catalytic RNA may involve generating separatepools of randomized ribozyme sequences, performing directed evolution onthe pools, screening the pools for desirable aminoacylation activity,and selecting sequences of those ribozymes exhibiting desiredaminoacylation activity.

Ribozymes can comprise motifs and/or regions that facilitate acylationactivity, such as a GGU motif and a U-rich region. For example, it hasbeen reported that U-rich regions can facilitate recognition of an aminoacid substrate, and a GGU-motif can form base pairs with the 3′ terminiof a tRNA. In combination, the GGU and motif and U-rich regionfacilitate simultaneous recognition of both the amino acid and tRNAsimultaneously, and thereby facilitate aminoacylation of the 3′ terminusof the tRNA.

Ribozymes can be generated by in vitro selection using a partiallyrandomized r24mini conjugated with tRNA^(Asn) _(CCCG), followed bysystematic engineering of a consensus sequence found in the activeclones. An exemplary ribozyme obtained by this method is termed “Fx3ribozyme” and is described in U.S. Pub. App. No. 2003/0228593, thecontents of which is incorporated by reference herein, acts as aversatile catalyst for the synthesis of various aminoacyl-tRNAs chargedwith cognate non-natural amino acids.

Immobilization on a substrate may be used to enable efficient affinitypurification of the aminoacylated tRNAs. Examples of suitable substratesinclude, but are not limited to, agarose, sepharose, and magnetic beads.Ribozymes can be immobilized on resins by taking advantage of thechemical structure of RNA, such as the 3′-cis-diol on the ribose of RNAcan be oxidized with periodate to yield the corresponding dialdehyde tofacilitate immobilization of the RNA on the resin. Various types ofresins can be used including inexpensive hydrazide resins whereinreductive amination makes the interaction between the resin and theribozyme an irreversible linkage. Synthesis of aminoacyl-tRNAs can besignificantly facilitated by this on-column aminoacylation technique.Kourouklis et al. Methods 2005; 36:239-4 describe a column-basedaminoacylation system.

Isolation of the aminoacylated tRNAs can be accomplished in a variety ofways. One suitable method is to elute the aminoacylated tRNAs from acolumn with a buffer such as a sodium acetate solution with 10 mM EDTA,a buffer containing 50 mMN-(2-hydroxyethyl)piperazine-N′-(3-propanesulfonic acid), 12.5 mM KCl,pH 7.0, 10 mM EDTA, or simply an EDTA buffered water (pH 7.0).

The aminoacylated tRNAs can be added to translation reactions in orderto incorporate the amino acid with which the tRNA was aminoacylated in aposition of choice in a polypeptide made by the translation reaction.Examples of translation systems in which the aminoacylated tRNAs of thepresent invention may be used include, but are not limited to celllysates. Cell lysates provide reaction components necessary for in vitrotranslation of a polypeptide from an input mRNA. Examples of suchreaction components include but are not limited to ribosomal proteins,rRNA, amino acids, tRNAs, GTP, ATP, translation initiation andelongation factors and additional factors associated with translation.Additionally, translation systems may be batch translations orcompartmentalized translation. Batch translation systems combinereaction components in a single compartment while compartmentalizedtranslation systems separate the translation reaction components fromreaction products that can inhibit the translation efficiency. Suchtranslation systems are available commercially.

Further, a coupled transcription/translation system may be used. Coupledtranscription/translation systems allow for both transcription of aninput DNA into a corresponding mRNA, which is in turn translated by thereaction components. An example of a commercially available coupledtranscription/translation is the Rapid Translation System (RTS, RocheInc.). The system includes a mixture containing E. coli lysate forproviding translational components such as ribosomes and translationfactors. Additionally, an RNA polymerase is included for thetranscription of the input DNA into an mRNA template for use intranslation. RTS can use compartmentalization of the reaction componentsby way of a membrane interposed between reaction compartments, includinga supply/waste compartment and a transcription/translation compartment.

Aminoacylation of tRNA may be performed by other agents, including butnot limited to, transferases, polymerases, catalytic antibodies,multi-functional proteins, and the like.

Lu et al. in Mol Cell. 2001 October; 8(4):759-69 describe a method inwhich a protein is chemically ligated to a synthetic peptide containingunnatural amino acids (expressed protein ligation).

Microinjection techniques have also been use incorporate unnatural aminoacids into proteins. See, e.g., M. W. Nowak, P. C. Kearney, J. R.Sampson, M. E. Saks, C. G. Labarca, S. K. Silverman, W. G. Zhong, J.Thorson, J. N. Abelson, N. Davidson, P. G. Schultz, D. A. Dougherty andH. A. Lester, Science, 268:439 (1995); and, D. A. Dougherty, Curr. Opin.Chem. Biol., 4:645 (2000). A Xenopus oocyte was coinjected with two RNAspecies made in vitro: an mRNA encoding the target protein with a UAGstop codon at the amino acid position of interest and an ambersuppressor tRNA aminoacylated with the desired unnatural amino acid. Thetranslational machinery of the oocyte then inserts the unnatural aminoacid at the position specified by UAG. This method has allowed in vivostructure-function studies of integral membrane proteins, which aregenerally not amenable to in vitro expression systems. Examples includethe incorporation of a fluorescent amino acid into tachykininneurokinin-2 receptor to measure distances by fluorescence resonanceenergy transfer, see, e.g., G. Turcatti, K. Nemeth, M. D. Edgerton, U.Meseth, F. Talabot, M. Peitsch, J. Knowles, H. Vogel and A. Chollet, J.Biol. Chem., 271:19991 (1996); the incorporation of biotinylated aminoacids to identify surface-exposed residues in ion channels, see, e.g.,J. P. Gallivan, H. A. Lester and D. A. Dougherty, Chem. Biol., 4:739(1997); the use of caged tyrosine analogs to monitor conformationalchanges in an ion channel in real time, see, e.g., J. C. Miller, S. K.Silverman, P. M. England, D. A. Dougherty and H. A. Lester, Neuron,20:619 (1998); and, the use of alpha hydroxy amino acids to change ionchannel backbones for probing their gating mechanisms. See, e.g., P. M.England, Y. Zhang, D. A. Dougherty and H. A. Lester, Cell, 96:89 (1999);and, T. Lu, A. Y. Ting, J. Mainland, L. Y. Jan, P. G. Schultz and J.Yang, Nat. Neurosci., 4:239 (2001).

The ability to incorporate unnatural amino acids directly into proteinsin vivo offers a wide variety of advantages including but not limitedto, high yields of mutant proteins, technical ease, the potential tostudy the mutant proteins in cells or possibly in living organisms andthe use of these mutant proteins in therapeutic treatments anddiagnostic uses. The ability to include unnatural amino acids withvarious sizes, acidities, nucleophilicities, hydrophobicities, and otherproperties into proteins can greatly expand our ability to rationallyand systematically manipulate the structures of proteins, both to probeprotein function and create new proteins or organisms with novelproperties.

In one attempt to site-specifically incorporate para-F-Phe, a yeastamber suppressor tRNAPheCUA/phenylalanyl-tRNA synthetase pair was usedin a p-F-Phe resistant, Phe auxotrophic Escherichia coli strain. See,e.g., R. Furter, Protein Sci., 7:419 (1998).

It may also be possible to obtain expression of a hGH polynucleotide ofthe present invention using a cell-free (in-vitro) translational system.Translation systems may be cellular or cell-free, and may be prokaryoticor eukaryotic. Cellular translation systems include, but are not limitedto, whole cell preparations such as permeabilized cells or cell cultureswherein a desired nucleic acid sequence can be transcribed to mRNA andthe mRNA translated. Cell-free translation systems are commerciallyavailable and many different types and systems are well-known. Examplesof cell-free systems include, but are not limited to, prokaryoticlysates such as Escherichia coli lysates, and eukaryotic lysates such aswheat germ extracts, insect cell lysates, rabbit reticulocyte lysates,rabbit oocyte lysates and human cell lysates. Eukaryotic extracts orlysates may be preferred when the resulting protein is glycosylated,phosphorylated or otherwise modified because many such modifications areonly possible in eukaryotic systems. Some of these extracts and lysatesare available commercially (Promega; Madison, Wis.; Stratagene; LaJolla, Calif.; Amersham; Arlington Heights, Ill.; GIBCO/BRL; GrandIsland, N.Y.). Membranous extracts, such as the canine pancreaticextracts containing microsomal membranes, are also available which areuseful for translating secretory proteins. In these systems, which caninclude either mRNA as a template (in-vitro translation) or DNA as atemplate (combined in-vitro transcription and translation), the in vitrosynthesis is directed by the ribosomes. Considerable effort has beenapplied to the development of cell-free protein expression systems. See,e.g., Kim, D. M. and J. R. Swartz, Biotechnology and Bioengineering,74:309-316 (2001); Kim, D. M. and J. R. Swartz, Biotechnology Letters,22, 1537-1542, (2000); Kim, D. M., and J. R. Swartz, BiotechnologyProgress, 16, 385-390, (2000); Kim, D. M., and J. R. Swartz,Biotechnology and Bioengineering, 66, 180-188, (1999); and Patnaik, R.and J. R. Swartz, Biotechniques 24, 862-868, (1998); U.S. Pat. No.6,337,191; U.S. Patent Publication No. 2002/0081660; WO 00/55353; WO90/05785, which are incorporated by reference herein. Another approachthat may be applied to the expression of hGH polypeptides comprising anon-naturally encoded amino acid includes the mRNA-peptide fusiontechnique. See, e.g., R. Roberts and J. Szostak, Proc. Natl. Acad. Sci.(USA) 94:12297-12302 (1997); A. Frankel, et al., Chemistry & Biology10:1043-105.0 (2003). In this approach, an mRNA template linked topuromycin is translated into peptide on the ribosome. If one or moretRNA molecules has been modified, non-natural amino acids can beincorporated into the peptide as well. After the last mRNA codon hasbeen read, puromycin captures the C-terminus of the peptide. If theresulting mRNA-peptide conjugate is found to have interesting propertiesin an in vitro assay, its identity can be easily revealed from the mRNAsequence. In this way, one may screen libraries of hGH polypeptidescomprising one or more non-naturally encoded amino acids to identifypolypeptides having desired properties. More recently, in vitro ribosometranslations with purified components have been reported that permit thesynthesis of peptides substituted with non-naturally encoded aminoacids. See, e.g., A. Forster et al., Proc. Natl. Acad. Sci. (USA)100:6353 (2003).

Reconstituted translation systems may also be used. Mixtures of purifiedtranslation factors have also been used successfully to translate mRNAinto protein as well as combinations of lysates or lysates supplementedwith purified translation factors such as initiation factor-1 (IF-1),IF-2, IF-3 (α or β), elongation factor T (EF-Tu), or terminationfactors. Cell-free systems may also be coupled transcription/translationsystems wherein DNA is introduced to the system, transcribed into mRNAand the mRNA translated as described in Current Protocols in MolecularBiology (F. M. Ausubel et al. editors, Wiley Interscience, 1993), whichis hereby specifically incorporated by reference. RNA transcribed ineukaryotic transcription system may be in the form of heteronuclear RNA(hnRNA) or 5′-end caps (7-methyl guanosine) and 3′-end poly A tailedmature mRNA, which can be an advantage in certain translation systems.For example, capped mRNAs are translated with high efficiency in thereticulocyte lysate system.

VIII. Macromolecular Polymers Coupled to hGH Polypeptides

Various modifications to the non-natural amino acid polypeptidesdescribed herein can be effected using the compositions, methods,techniques and strategies described herein. These modifications includethe incorporation of further functionality onto the non-natural aminoacid component of the polypeptide, including but not limited to, alabel; a dye; a polymer; a water-soluble polymer; a derivative ofpolyethylene glycol; a photocrosslinker; a radionuclide; a cytotoxiccompound; a drug; an affinity label; a photoaffinity label; a reactivecompound; a resin; a second protein or polypeptide or polypeptideanalog; an antibody or antibody fragment; a metal chelator; a cofactor;a fatty acid; a carbohydrate; a polynucleotide; a DNA; a RNA; anantisense polynucleotide; a saccharide; a water-soluble dendrimer; acyclodextrin; an inhibitory ribonucleic acid; a biomaterial; ananoparticle; a spin label; a fluorophore, a metal-containing moiety; aradioactive moiety; a novel functional group; a group that covalently ornoncovalently interacts with other molecules; a photocaged moiety; anactinic radiation excitable moiety; a photoisomerizable moiety; biotin;a derivative of biotin; a biotin analogue; a moiety incorporating aheavy atom; a chemically cleavable group; a photocleavable group; anelongated side chain; a carbon-linked sugar; a redox-active agent; anamino thioacid; a toxic moiety; an isotopically labeled moiety; abiophysical probe; a phosphorescent group; a chemiluminescent group; anelectron dense group; a magnetic group; an intercalating group; achromophore; an energy transfer agent; a biologically active agent; adetectable label; a small molecule; a quantum dot; a nanotransmitter; aradionucleotide; a radiotransmitter; a neutron-capture agent; or anycombination of the above, or any other desirable compound or substance.As an illustrative, non-limiting example of the compositions, methods,techniques and strategies described herein, the following descriptionwill focus on adding macromolecular polymers to the non-natural aminoacid polypeptide with the understanding that the compositions, methods,techniques and strategies described thereto are also applicable (withappropriate modifications, if necessary and for which one of skill inthe art could make with the disclosures herein) to adding otherfunctionalities, including but not limited to those listed above.

A wide variety of macromolecular polymers and other molecules can belinked to hGH polypeptides of the present invention to modulatebiological properties of the hGH polypeptide, and/or provide newbiological properties to the hGH molecule. These macromolecular polymerscan be linked to the hGH polypeptide via a naturally encoded amino acid,via a non-naturally encoded amino acid, or any functional substituent ofa natural or non-natural amino acid, or any substituent or functionalgroup added to a natural or non-natural amino acid. The molecular weightof the polymer may be of a wide range, including but not limited to,between about 100 Da and about 100,000 Da or more. The molecular weightof the polymer may be between about 100 Da and about 100,000 Da,including but not limited to, 100,000 Da, 95,000 Da, 90,000 Da, 85,000Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da,50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000Da, 4,000 Da, 3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600Da, 500 Da, 400 Da, 300 Da, 200 Da, and 100 Da. In some embodiments, themolecular weight of the polymer is between about 100 Da and 50,000 Da.In some embodiments, the molecular weight of the polymer is betweenabout 100 Da and 40,000 Da. In some embodiments, the molecular weight ofthe polymer is between about 1,000 Da and 40,000 Da. In someembodiments, the molecular weight of the polymer is between about 5,000Da and 40,000 Da. In some embodiments, the molecular weight of thepolymer is between about 10,000 Da and 40,000 Da.

The present invention provides substantially homogenous preparations ofpolymer:protein conjugates. “Substantially homogenous” as used hereinmeans that polymer:protein conjugate molecules are observed to begreater than half of the total protein. The polymer:protein conjugatehas biological activity and the present “substantially homogenous”PEGylated hGH polypeptide preparations provided herein are those whichare homogenous enough to display the advantages of a homogenouspreparation, e.g., ease in clinical application in predictability of lotto lot pharmacokinetics.

One may also choose to prepare a mixture of polymer:protein conjugatemolecules, and the advantage provided herein is that one may select theproportion of mono-polymer:protein conjugate to include in the mixture.Thus, if desired, one may prepare a mixture of various proteins withvarious numbers of polymer moieties attached (i.e., di-, tri-, tetra-,etc.) and combine said conjugates with the mono-polymer:proteinconjugate prepared using the methods of the present invention, and havea mixture with a predetermined proportion of mono-polymer:proteinconjugates.

The polymer selected may be water soluble so that the protein to whichit is attached does not precipitate in an aqueous environment, such as aphysiological environment. The polymer may be branched or unbranched.For therapeutic use of the end-product preparation, the polymer will bepharmaceutically acceptable.

The proportion of polyethylene glycol molecules to protein moleculeswill vary, as will their concentrations in the reaction mixture. Ingeneral, the optimum ratio (in terms of efficiency of reaction in thatthere is minimal excess unreacted protein or polymer) may be determinedby the molecular weight of the polyethylene glycol selected and on thenumber of available reactive groups available. As relates to molecularweight, typically the higher the molecular weight of the polymer, thefewer number of polymer molecules which may be attached to the protein.Similarly, branching of the polymer should be taken into account whenoptimizing these parameters. Generally, the higher the molecular weight(or the more branches) the higher the polymer:protein ratio.

Examples of polymers include but are not limited to polyalkyl ethers andalkoxy-capped analogs thereof (e.g., polyoxyethylene glycol,polyoxyethylene/propylene glycol, and methoxy or ethoxy-capped analogsthereof, especially polyoxyethylene glycol, the latter is also known aspolyethyleneglycol or PEG); polyvinylpyrrolidones; polyvinylalkylethers; polyoxazolines, polyalkyl oxazolines and polyhydroxyalkyloxazolines; polyacrylamides, polyalkyl acrylamides, and polyhydroxyalkylacrylamides (e.g., polyhydroxypropylmethacrylamide and derivativesthereof); polyhydroxyalkyl acrylates; polysialic acids and analogsthereof; hydrophilic peptide sequences; polysaccharides and theirderivatives, including dextran and dextran derivatives, e.g.,carboxymethyldextran, dextran sulfates, aminodextran; cellulose and itsderivatives, e.g., carboxymethyl cellulose, hydroxyalkyl celluloses;chitin and its derivatives, e.g., chitosan, succinyl chitosan,carboxymethylchitin, carboxymethylchitosan; hyaluronic acid and itsderivatives; starches; alginates; chondroitin sulfate; albumin; pullulanand carboxymethyl pullulan; polyaminoacids and derivatives thereof,e.g., polyglutamic acids, polylysines, polyaspartic acids,polyaspartamides; maleic anhydride copolymers such as: styrene maleicanhydride copolymer, divinylethyl ether maleic anhydride copolymer;polyvinyl alcohols; copolymers thereof; terpolymers thereof; mixturesthereof; and derivatives of the foregoing.

The proportion of polyethylene glycol molecules to protein moleculeswill vary, as will their concentrations in the reaction mixture. Ingeneral, the optimum ratio (in terms of efficiency of reaction in thatthere is minimal excess unreacted protein or polymer) may be determinedby the molecular weight of the polyethylene glycol selected and on thenumber of available reactive groups available. As relates to molecularweight, typically the higher the molecular weight of the polymer, thefewer number of polymer molecules which may be attached to the protein.Similarly, branching of the polymer should be taken into account whenoptimizing these parameters. Generally, the higher the molecular weight(or the more branches) the higher the polymer:protein ratio.

As used herein, and when contemplating PEG:hGH polypeptide conjugates,the term “therapeutically effective amount” refers to an amount whichgives the desired benefit to a patient. The amount will vary from oneindividual to another and will depend upon a number of factors,including the overall physical condition of the patient and theunderlying cause of the condition to be treated. The amount of hGHpolypeptide used for therapy gives an acceptable rate of change andmaintains the desired change at a beneficial level. A therapeuticallyeffective amount of the present compositions may be readily ascertainedby one of ordinary skill in the art using publicly available materialsand procedures.

The water soluble polymer may be any structural form including but notlimited to linear, forked or branched. Typically, the water solublepolymer is a poly(alkylene glycol), such as poly(ethylene glycol) (PEG),but other water soluble polymers can also be employed. By way ofexample, PEG is used to describe certain embodiments of this invention.

PEG is a well-known, water soluble polymer that is commerciallyavailable or can be prepared by ring-opening polymerization of ethyleneglycol according to methods known to those of ordinary skill in the art(Sandler and Karo, Polymer Synthesis, Academic Press, New York, Vol. 3,pages 138-161). The term “PEG” is used broadly to encompass anypolyethylene glycol molecule, without regard to size or to modificationat an end of the PEG, and can be represented as linked to the hGHpolypeptide by the formula:

XO—(CH₂CH₂O)_(n)—CH₂CH₂—Y

where n is 2 to 10,000 and X is H or a terminal modification, includingbut not limited to, a C₁₋₄ alkyl, a protecting group, or a terminalfunctional group.

In some cases, a PEG used in the invention terminates on one end withhydroxy or methoxy, i.e., X is H or CH₃ (“methoxy PEG”). Alternatively,the PEG can terminate with a reactive group, thereby forming abifunctional polymer. Typical reactive groups can include those reactivegroups that are commonly used to react with the functional groups foundin the 20 common amino acids (including but not limited to, maleimidegroups, activated carbonates (including but not limited to,p-nitrophenyl ester), activated esters (including but not limited to,N-hydroxysuccinimide, p-nitrophenyl ester) and aldehydes) as well asfunctional groups that are inert to the 20 common amino acids but thatreact specifically with complementary functional groups present innon-naturally encoded amino acids (including but not limited to, azidegroups, alkyne groups). It is noted that the other end of the PEG, whichis shown in the above formula by Y, will attach either directly orindirectly to a hGH polypeptide via a naturally-occurring ornon-naturally encoded amino acid. For instance, Y may be an amide,carbamate or urea linkage to an amine group (including but not limitedto, the epsilon amine of lysine or the N-terminus) of the polypeptide.Alternatively, Y may be a maleimide linkage to a thiol group (includingbut not limited to, the thiol group of cysteine). Alternatively, Y maybe a linkage to a residue not commonly accessible via the 20 commonamino acids. For example, an azide group on the PEG can be reacted withan alkyne group on the hGH polypeptide to form a Huisgen[3+2]cycloaddition product. Alternatively, an alkyne group on the PEGcan be reacted with an azide group present in a non-naturally encodedamino acid to form a similar product. In some embodiments, a strongnucleophile (including but not limited to, hydrazine, hydrazide,hydroxylamine, semicarbazide) can be reacted with an aldehyde or ketonegroup present in a non-naturally encoded amino acid to form a hydrazone,oxime or semicarbazone, as applicable, which in some cases can befurther reduced by treatment with an appropriate reducing agent.Alternatively, the strong nucleophile can be incorporated into the hGHpolypeptide via a non-naturally encoded amino acid and used to reactpreferentially with a ketone or aldehyde group present in the watersoluble polymer.

Any molecular mass for a PEG can be used as practically desired,including but not limited to, from about 100 Daltons (Da) to 100,000 Daor more as desired (including but not limited to, sometimes 0.1-50 kDaor 10-40 kDa). The molecular weight of the PEG may be of a wide range,including but not limited to, between about 100 Da and about 100,000 Daor more. The molecular weight of the PEG may be between about 100 Da andabout 100,000 Da, including but not limited to, 100,000 Da, 95,000 Da,90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da,25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, 1,000 Da, 900 Da,800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, and 100 Da. Insome embodiments, the molecular weight of the PEG is between about 100Da and 50,000 Da. In some embodiments, the molecular weight of the PEGis between about 100 Da and 40,000 Da. In some embodiments, themolecular weight of the PEG is between about 1,000 Da and 40,000 Da. Insome embodiments, the molecular weight of the PEG is between about 5,000Da and 40,000 Da. In some embodiments, the molecular weight of the PEGis between about 10,000 Da and 40,000 Da. Branched chain PEGs, includingbut not limited to, PEG molecules with each chain having a MW rangingfrom 1-100 kDa (including but not limited to, 1-50 kDa or 5-20 kDa) canalso be used. The molecular weight of the branched chain PEG may be,including but not limited to, between about 1,000 Da and about 100,000Da or more. The molecular weight of the branched chain PEG may bebetween about 1,000 Da and about 100,000 Da, including but not limitedto, 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da,70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da,9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da,2,000 Da, and 1,000 Da. In some embodiments, the molecular weight of thebranched chain PEG is between about 1,000 Da and 50,000 Da. In someembodiments, the molecular weight of the branched chain PEG is betweenabout 1,000 Da and 40,000 Da. In some embodiments, the molecular weightof the branched chain PEG is between about 5,000 Da and 40,000 Da. Insome embodiments, the molecular weight of the branched chain PEG isbetween about 5,000 Da and 20,000 Da. A wide range of PEG molecules aredescribed in, including but not limited to, the Shearwater Polymers,Inc. catalog, Nektar Therapeutics catalog, incorporated herein byreference.

Generally, at least one terminus of the PEG molecule is available forreaction with the non-naturally-encoded amino acid. For example, PEGderivatives bearing alkyne and azide moieties for reaction with aminoacid side chains can be used to attach PEG to non-naturally encodedamino acid. If the non-naturally encoded amino acid comprises an azide,then the PEG will typically contain either an alkyne moiety to effectformation of the [3+2]cycloaddition product or an activated PEG species(i.e., ester, carbonate) containing a phosphine group to effectformation of the amide linkage. Alternatively, if the non-naturallyencoded amino acid comprises an alkyne, then the PEG will typicallycontain an azide moiety to effect formation of the [3+2] Huisgencycloaddition product. If the non-naturally encoded amino acid comprisesa carbonyl group, the PEG will typically comprise a potent nucleophile(including but not limited to, a hydrazide, hydrazine, hydroxylamine, orsemicarbazide functionality) in order to effect formation ofcorresponding hydrazone, oxime, and semicarbazone linkages,respectively. In other alternatives, a reverse of the orientation of thereactive groups described above can be used, i.e., an azide moiety inthe non-naturally encoded amino acid can be reacted with a PEGderivative containing an alkyne.

In some embodiments, the hGH polypeptide variant with a PEG derivativecontains a chemical functionality that is reactive with the chemicalfunctionality present on the side chain of the non-naturally encodedamino acid.

The invention provides in some embodiments azide- andacetylene-containing polymer derivatives comprising a water solublepolymer backbone having an average molecular weight from about 800 Da toabout 100,000 Da. The polymer backbone of the water-soluble polymer canbe poly(ethylene glycol). However, it should be understood that a widevariety of water soluble polymers including but not limited topoly(ethylene)glycol and other related polymers, including poly(dextran)and poly(propylene glycol), are also suitable for use in the practice ofthis invention and that the use of the term PEG or poly(ethylene glycol)is intended to encompass and include all such molecules. The term PEGincludes, but is not limited to, poly(ethylene glycol) in any of itsforms, including bifunctional PEG, multiarmed PEG, derivatized PEG,forked PEG, branched PEG, pendent PEG (i.e. PEG or related polymershaving one or more functional groups pendent to the polymer backbone),or PEG with degradable linkages therein.

PEG is typically clear, colorless, odorless, soluble in water, stable toheat, inert to many chemical agents, does not hydrolyze or deteriorate,and is generally non-toxic. Poly(ethylene glycol) is considered to bebiocompatible, which is to say that PEG is capable of coexistence withliving tissues or organisms without causing harm. More specifically, PEGis substantially non-immunogenic, which is to say that PEG does not tendto produce an immune response in the body. When attached to a moleculehaving some desirable function in the body, such as a biologicallyactive agent, the PEG tends to mask the agent and can reduce oreliminate any immune response so that an organism can tolerate thepresence of the agent. PEG conjugates tend not to produce a substantialimmune response or cause clotting or other undesirable effects. PEGhaving the formula —CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—, where n is from about3 to about 4000, typically from about 20 to about 2000, is suitable foruse in the present invention. PEG having a molecular weight of fromabout 800 Da to about 100,000 Da are in some embodiments of the presentinvention particularly useful as the polymer backbone. The molecularweight of PEG may be of a wide range, including but not limited to,between about 100 Da and about 100,000 Da or more. The molecular weightof PEG may be between about 100 Da and about 100,000 Da, including butnot limited to, 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da,75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da,10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da,3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da,400 Da, 300 Da, 200 Da, and 100 Da. In some embodiments, the molecularweight of PEG is between about 100 Da and 50,000 Da. In someembodiments, the molecular weight of PEG is between about 100 Da and40,000 Da. In some embodiments, the molecular weight of PEG is betweenabout 1,000 Da and 40,000 Da. In some embodiments, the molecular weightof PEG is between about 5,000 Da and 40,000 Da. In some embodiments, themolecular weight of PEG is between about 10,000 Da and 40,000 Da.

The polymer backbone can be linear or branched. Branched polymerbackbones are generally known in the art. Typically, a branched polymerhas a central branch core moiety and a plurality of linear polymerchains linked to the central branch core. PEG is commonly used inbranched forms that can be prepared by addition of ethylene oxide tovarious polyols, such as glycerol, glycerol oligomers, pentaerythritoland sorbitol. The central branch moiety can also be derived from severalamino acids, such as lysine. The branched poly(ethylene glycol) can berepresented in general form as R(—PEG-OH)_(m) in which R is derived froma core moiety, such as glycerol, glycerol oligomers, or pentaerythritol,and m represents the number of arms. Multi-armed PEG molecules, such asthose described in U.S. Pat. Nos. 5,932,462 5,643,575; 5,229,490;4,289,872; U.S. Pat. Appl. 2003/0143596; WO 96/21469; and WO 93/21259,each of which is incorporated by reference herein in its entirety, canalso be used as the polymer backbone.

Branched PEG can also be in the form of a forked PEG represented byPEG(-YCHZ₂)_(n), where Y is a linking group and Z is an activatedterminal group linked to CH by a chain of atoms of defined length.

Yet another branched form, the pendant PEG, has reactive groups, such ascarboxyl, along the PEG backbone rather than at the end of PEG chains.

In addition to these forms of PEG, the polymer can also be prepared withweak or degradable linkages in the backbone. For example, PEG can beprepared with ester linkages in the polymer backbone that are subject tohydrolysis. As shown below, this hydrolysis results in cleavage of thepolymer into fragments of lower molecular weight:

-PEG-CO₂-PEG-+H₂O→PEG-CO₂H+HO-PEG-

It is understood by those of ordinary skill in the art that the termpoly(ethylene glycol) or PEG represents or includes all the forms knownin the art including but not limited to those disclosed herein.

Many other polymers are also suitable for use in the present invention.In some embodiments, polymer backbones that are water-soluble, with from2 to about 300 termini, are particularly useful in the invention.Examples of suitable polymers include, but are not limited to, otherpoly(alkylene glycols), such as poly(propylene glycol) (“PPG”),copolymers thereof (including but not limited to copolymers of ethyleneglycol and propylene glycol), terpolymers thereof, mixtures thereof, andthe like. Although the molecular weight of each chain of the polymerbackbone can vary, it is typically in the range of from about 800 Da toabout 100,000 Da, often from about 6,000 Da to about 80,000 Da. Themolecular weight of each chain of the polymer backbone may be betweenabout 100 Da and about 100,000 Da, including but not limited to, 100,000Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da,65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da,8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da,1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200Da, and 100 Da. In some embodiments, the molecular weight of each chainof the polymer backbone is between about 100 Da and 50,000 Da. In someembodiments, the molecular weight of each chain of the polymer backboneis between about 100 Da and 40,000 Da. In some embodiments, themolecular weight of each chain of the polymer backbone is between about1,000 Da and 40,000 Da. In some embodiments, the molecular weight ofeach chain of the polymer backbone is between about 5,000 Da and 40,000Da. In some embodiments, the molecular weight of each chain of thepolymer backbone is between about 10,000 Da and 40,000 Da.

Those of ordinary skill in the art will recognize that the foregoinglist for substantially water soluble backbones is by no means exhaustiveand is merely illustrative, and that all polymeric materials having thequalities described above are contemplated as being suitable for use inthe present invention.

In some embodiments of the present invention the polymer derivatives are“multi-functional”, meaning that the polymer backbone has at least twotermini, and possibly as many as about 300 termini, functionalized oractivated with a functional group. Multifunctional polymer derivativesinclude, but are not limited to, linear polymers having two termini,each terminus being bonded to a functional group which may be the sameor different.

In one embodiment, the polymer derivative has the structure:

X-A-POLY-B—N═N═N

wherein:N═N═N is an azide moiety;B is a linking moiety, which may be present or absent;POLY is a water-soluble non-antigenic polymer;A is a linking moiety, which may be present or absent and which may bethe same as B or different; andX is a second functional group.Examples of a linking moiety for A and B include, but are not limitedto, a multiply-functionalized alkyl group containing up to 18, includingbut not limited to, between 1-10 carbon atoms. A heteroatom such asnitrogen, oxygen or sulfur may be included with the alkyl chain. Thealkyl chain may also be branched at a heteroatom. Other examples of alinking moiety for A and B include, but are not limited to, a multiplyfunctionalized aryl group, containing up to 10, including but notlimited to, 5-6 carbon atoms. The aryl group may be substituted with onemore carbon atoms, nitrogen, oxygen or sulfur atoms. Other examples ofsuitable linking groups include those linking groups described in U.S.Pat. Nos. 5,932,462; 5,643,575; and U.S. Pat. Appl. Publication2003/0143596, each of which is incorporated by reference herein. Thoseof ordinary skill in the art will recognize that the foregoing list forlinking moieties is by no means exhaustive and is merely illustrative,and that all linking moieties having the qualities described above arecontemplated to be suitable for use in the present invention.

Examples of suitable functional groups for use as X include, but are notlimited to, hydroxyl, protected hydroxyl, alkoxyl, active ester, such asN-hydroxysuccinimidyl esters and 1-benzotriazolyl esters, activecarbonate, such as N-hydroxysuccinimidyl carbonates and 1-benzotriazolylcarbonates, acetal, aldehyde, aldehyde hydrates, alkenyl, acrylate,methacrylate, acrylamide, active sulfone, amine, aminooxy, protectedamine, hydrazide, protected hydrazide, protected thiol, carboxylic acid,protected carboxylic acid, isocyanate, isothiocyanate, maleimide,vinylsulfone, dithiopyridine, vinylpyridine, iodoacetamide, epoxide,glyoxals, diones, mesylates, tosylates, tresylate, alkene, ketone, andazide. As is understood by those of ordinary skill in the art, theselected X moiety should be compatible with the azide group so thatreaction with the azide group does not occur. The azide-containingpolymer derivatives may be homobifunctional, meaning that the secondfunctional group (i.e., X) is also an azide moiety, orheterobifunctional, meaning that the second functional group is adifferent functional group.

The term “protected” refers to the presence of a protecting group ormoiety that prevents reaction of the chemically reactive functionalgroup under certain reaction conditions. The protecting group will varydepending on the type of chemically reactive group being protected. Forexample, if the chemically reactive group is an amine or a hydrazide,the protecting group can be selected from the group oftert-butyloxycarbonyl (t-Boc) and 9-fluorenylmethoxycarbonyl (Fmoc). Ifthe chemically reactive group is a thiol, the protecting group can beorthopyridyldisulfide. If the chemically reactive group is a carboxylicacid, such as butanoic or propionic acid, or a hydroxyl group, theprotecting group can be benzyl or an alkyl group such as methyl, ethyl,or tert-butyl. Other protecting groups known in the art may also be usedin the present invention.

Purification of the crude product can usually be accomplished by methodsknown in the art including, but are not limited to, precipitation of theproduct followed by chromatography, if necessary.

Water soluble polymers can be linked to the hGH polypeptides of theinvention. The water soluble polymers may be linked via a non-naturallyencoded amino acid incorporated in the hGH polypeptide or any functionalgroup or substituent of a non-naturally encoded or naturally encodedamino acid, or any functional group or substituent added to anon-naturally encoded or naturally encoded amino acid. Alternatively,the water soluble polymers are linked to a hGH polypeptide incorporatinga non-naturally encoded amino acid via a naturally-occurring amino acid(including but not limited to, cysteine, lysine or the amine group ofthe N-terminal residue). In some cases, the hGH polypeptides of theinvention comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 non-natural aminoacids, wherein one or more non-naturally-encoded amino acid(s) arelinked to water soluble polymer(s) (including but not limited to, PEGand/or oligosaccharides). In some cases, the hGH polypeptides of theinvention further comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or morenaturally-encoded amino acid(s) linked to water soluble polymers. Insome cases, the hGH polypeptides of the invention comprise one or morenon-naturally encoded amino acid(s) linked to water soluble polymers andone or more naturally-occurring amino acids linked to water solublepolymers. In some embodiments, the water soluble polymers used in thepresent invention enhance the serum half-life of the hGH polypeptiderelative to the unconjugated form.

The number of water soluble polymers linked to a hGH polypeptide (i.e.,the extent of PEGylation or glycosylation) of the present invention canbe adjusted to provide an altered (including but not limited to,increased or decreased) pharmacologic, pharmacokinetic orpharmacodynamic characteristic such as in vivo half-life. In someembodiments, the half-life of hGH is increased at least about 10, 20,30, 40, 50, 60, 70, 80, 90 percent, 2-fold, 5-fold, 6-fold, 7-fold,8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold,16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 25-fold, 30-fold, 35-fold,40-fold, 50-fold, or at least about 100-fold over an unmodifiedpolypeptide.

PEG Derivatives Containing a Strong Nucleophilic Group (i.e., Hydrazide,Hydrazine, Hydroxylamine or Semicarbazide)

In one embodiment of the present invention, a hGH polypeptide comprisinga carbonyl-containing non-naturally encoded amino acid is modified witha PEG derivative that contains a terminal hydrazine, hydroxylamine,hydrazide or semicarbazide moiety that is linked directly to the PEGbackbone.

In some embodiments, the hydroxylamine-terminal PEG derivative will havethe structure:

RO—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—O—NH₂

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000 (i.e., average molecular weight is between 5-40 kDa).

In some embodiments, the hydrazine- or hydrazide-containing PEGderivative will have the structure:

RO—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—X—NH—NH₂

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000 and X is optionally a carbonyl group (C═O) that can bepresent or absent.

In some embodiments, the semicarbazide-containing PEG derivative willhave the structure:

RO—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—NH—C(O)—NH—NH₂

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000.

In another embodiment of the invention, a hGH polypeptide comprising acarbonyl-containing amino acid is modified with a PEG derivative thatcontains a terminal hydroxylamine, hydrazide, hydrazine, orsemicarbazide moiety that is linked to the PEG backbone by means of anamide linkage.

In some embodiments, the hydroxylamine-terminal PEG derivatives have thestructure:

RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—NH—C(O)(CH₂)_(m)—O—NH₂

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000 (i.e., average molecular weight is between 5-40 kDa).

In some embodiments, the hydrazine- or hydrazide-containing PEGderivatives have the structure:

RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—NH—C(O)(CH₂)_(m)—X—NH—NH₂

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10, n is100-1,000 and X is optionally a carbonyl group (C═O) that can be presentor absent.

In some embodiments, the semicarbazide-containing PEG derivatives havethe structure:

RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—NH—C(O)(CH₂)_(m)—NH—C(O)—NH—NH₂

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000.

In another embodiment of the invention, a hGH polypeptide comprising acarbonyl-containing amino acid is modified with a branched PEGderivative that contains a terminal hydrazine, hydroxylamine, hydrazideor semicarbazide moiety, with each chain of the branched PEG having a MWranging from 10-40 kDa. Each chain of the branched PEG may have a MWranging from 5-20 kDa.

In another embodiment of the invention, a hGH polypeptide comprising anon-naturally encoded amino acid is modified with a PEG derivativehaving a branched structure. For instance, in some embodiments, thehydrazine- or hydrazide-terminal PEG derivative will have the followingstructure:

[RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—NH—C(O)]₂CH(CH₂)_(m)—X—NH—NH₂

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000, and X is optionally a carbonyl group (C═O) that can bepresent or absent.

In some embodiments, the PEG derivatives containing a semicarbazidegroup will have the structure:

[RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—C(O)—NH—CH₂—CH₂]₂CH—X—(CH₂)_(m)—NH—C(O)—NH—NH₂

where R is a simple alkyl (methyl, ethyl, propyl, etc.), X is optionallyNH, O, S, C(O) or not present, m is 2-10 and n is 100-1,000.

In some embodiments, the PEG derivatives containing a hydroxylaminegroup will have the structure:

[RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—C(O)—NH—CH₂—CH₂]₂CH—X—(CH₂)_(m)—O—NH₂

where R is a simple alkyl (methyl, ethyl, propyl, etc.), X is optionallyNH, O, S, C(O) or not present, m is 2-10 and n is 100-1,000.

The degree and sites at which the water soluble polymer(s) are linked tothe hGH polypeptide can modulate the binding of the hGH polypeptide tothe hGH polypeptide receptor at Site 1. In some embodiments, thelinkages are arranged such that the hGH polypeptide binds the hGHpolypeptide receptor at Site 1 with a K_(d) of about 400 nM or lower,with a K_(d) of 150 nM or lower, and in some cases with a K_(d) of 100nM or lower, as measured by an equilibrium binding assay, such as thatdescribed in Spencer et al., J. Biol. Chem., 263:7862-7867 (1988) forhGH.

Methods and chemistry for activation of polymers as well as forconjugation of peptides are described in the literature and are known inthe art. Commonly used methods for activation of polymers include, butare not limited to, activation of functional groups with cyanogenbromide, periodate, glutaraldehyde, biepoxides, epichlorohydrin,divinylsulfone, carbodiimide, sulfonyl halides, trichlorotriazine, etc.(see, R. F. Taylor, (1991), PROTEIN IMMOBILISATION. FUNDAMENTAL ANDAPPLICATIONS, Marcel Dekker, N.Y.; S. S. Wong, (1992), CHEMISTRY OFPROTEIN CONJUGATION AND CROSSLINKING, CRC Press, Boca Raton; G. T.Hermanson et al., (1993), IMMOBILIZED AFFINITY LIGAND TECHNIQUES,Academic Press, N.Y.; Dunn, R. L., et al., Eds. POLYMERIC DRUGS AND DRUGDELIVERY SYSTEMS, ACS Symposium Series Vol. 469, American ChemicalSociety, Washington, D.C. 1991).

Several reviews and monographs on the functionalization and conjugationof PEG are available. See, for example, Harris, Macromol. Chem. Phys.C25: 325-373 (1985); Scouten, Methods in Enzymology 135: 30-65 (1987);Wong et al., Enzyme Microb. Technol. 14: 866-874 (1992); Delgado et al.,Critical Reviews in Therapeutic Drug Carrier Systems 9: 249-304 (1992);Zalipsky, Bioconjugate Chem. 6: 150-165 (1995).

Methods for activation of polymers can also be found in WO 94/17039,U.S. Pat. No. 5,324,844, WO 94/18247, WO 94/04193, U.S. Pat. No.5,219,564, U.S. Pat. No. 5,122,614, WO 90/13540, U.S. Pat. No.5,281,698, and WO 93/15189, and for conjugation between activatedpolymers and enzymes including but not limited to Coagulation FactorVIII (WO 94/15625), hemoglobin (WO 94/09027), oxygen carrying molecule(U.S. Pat. No. 4,412,989), ribonuclease and superoxide dismutase(Veronese at al., App. Biochem. Biotech. 11: 141-52 (1985)). Allreferences and patents cited are incorporated by reference herein.

PEGylation (i.e., addition of any water soluble polymer) of hGHpolypeptides containing a non-naturally encoded amino acid, such asp-azido-L-phenylalanine, is carried out by any convenient method. Forexample, hGH polypeptide is PEGylated with an alkyne-terminated mPEGderivative. Briefly, an excess of solid mPEG(5000)-O—CH₂—C≡CH is added,with stirring, to an aqueous solution of p-azido-L-Phe-containing hGHpolypeptide at room temperature. Typically, the aqueous solution isbuffered with a buffer having a pK_(a) near the pH at which the reactionis to be carried out (generally about pH 4-10). Examples of suitablebuffers for PEGylation at pH 7.5, for instance, include, but are notlimited to, HEPES, phosphate, borate, TRIS-HCl, EPPS, and TES. The pH iscontinuously monitored and adjusted if necessary. The reaction istypically allowed to continue for between about 1-48 hours.

The reaction products are subsequently subjected to hydrophobicinteraction chromatography to separate the PEGylated hGH polypeptidevariants from free mPEG(5000)-O—CH₂—C≡CH and any high-molecular weightcomplexes of the PEGylated hGH polypeptide which may form when unblockedPEG is activated at both ends of the molecule, thereby crosslinking hGHpolypeptide variant molecules. The conditions during hydrophobicinteraction chromatography are such that free mPEG(5000)-O—CH₂—C≡CHflows through the column, while any crosslinked PEGylated hGHpolypeptide variant complexes elute after the desired forms, whichcontain one hGH polypeptide variant molecule conjugated to one or morePEG groups. Suitable conditions vary depending on the relative sizes ofthe cross-linked complexes versus the desired conjugates and are readilydetermined by those of ordinary skill in the art. The eluent containingthe desired conjugates is concentrated by ultrafiltration and desaltedby diafiltration.

If necessary, the PEGylated hGH polypeptide obtained from thehydrophobic chromatography can be purified further by one or moreprocedures known to those of ordinary skill in the art including, butare not limited to, affinity chromatography; anion- or cation-exchangechromatography (using, including but not limited to, DEAE SEPHAROSE);chromatography on silica; reverse phase HPLC; gel filtration (using,including but not limited to, SEPHADEX G-75); hydrophobic interactionchromatography; size-exclusion chromatography, metal-chelatechromatography; ultrafiltration/diafiltration; ethanol precipitation;ammonium sulfate precipitation; chromatofocusing; displacementchromatography; electrophoretic procedures (including but not limited topreparative isoelectric focusing), differential solubility (includingbut not limited to ammonium sulfate precipitation), or extraction.Apparent molecular weight may be estimated by GPC by comparison toglobular protein standards (Preneta, A Z in PROTEIN PURIFICATIONMETHODS, A PRACTICAL APPROACH (Harris & Angal, Eds.) IRL Press 1989,293-306). The purity of the hGH-PEG conjugate can be assessed byproteolytic degradation (including but not limited to, trypsin cleavage)followed by mass spectrometry analysis. Pepinsky R B, et al., J.Pharmcol. & Exp. Ther. 297(3):1059-66 (2001).

A water soluble polymer linked to an amino acid of a hGH polypeptide ofthe invention can be further derivatized or substituted withoutlimitation.

Other PEG Derivatives and General PEGylation Techniques

Azide-containing, alkyne-containing, and phosphine-containing PEGderivatives are described in U.S. patent application Ser. No.11/046,432, entitled “Modified Human Growth Hormone Polypeptides andTheir Uses”.

Other exemplary PEG molecules that may be linked to hGH polypeptides, aswell as PEGylation methods include those described in, e.g., U.S. PatentPublication No. 2004/0001838; 2002/0052009; 2003/0162949; 2004/0013637;2003/0228274; 2003/0220447; 2003/0158333; 2003/0143596; 2003/0114647;2003/0105275; 2003/0105224; 2003/0023023; 2002/0156047; 2002/0099133;2002/0086939; 2002/0082345; 2002/0072573; 2002/0052430; 2002/0040076;2002/0037949; 2002/0002250; 2001/0056171; 2001/0044526; 2001/0021763;U.S. Pat. Nos. 6,646,110; 5,824,778; 5,476,653; 5,219,564; 5,629,384;5,736,625; 4,902,502; 5,281,698; 5,122,614; 5,473,034; 5,516,673;5,382,657; 6,552,167; 6,610,281; 6,515,100; 6,461,603; 6,436,386;6,214,966; 5,990,237; 5,900,461; 5,739,208; 5,672,662; 5,446,090;5,808,096; 5,612,460; 5,324,844; 5,252,714; 6,420,339; 6,201,072;6,451,346; 6,306,821; 5,559,213; 5,747,646; 5,834,594; 5,849,860;5,980,948; 6,004,573; 6,129,912; WO 97/32607, EP 229,108, EP 402,378, WO92/16555, WO 94/04193, WO 94/14758, WO 94/17039, WO 94/18247, WO94/28024, WO 95/00162, WO 95/11924, WO95/13090, WO 95/33490, WO96/00080, WO 97/18832, WO 98/41562, WO 98/48837, WO 99/32134, WO99/32139, WO 99/32140, WO 96/40791, WO 98/32466, WO 95/06058, EP 439508, WO 97/03106, WO 96/21469, WO 95/13312, EP 921 131, WO 98/05363, EP809 996, WO 96/41813, WO 96/07670, EP 605 963, EP 510 356, EP 400 472,EP 183 503 and EP 154 316, which are incorporated by reference herein.Any of the PEG molecules described herein may be used in any form,including but not limited to, single chain, branched chain, multiarmchain, single functional, bi-functional, multi-functional, or anycombination thereof. U.S. patent application Ser. No. 11/046,432entitled “Modified Human Growth Hormone Polypeptides and Their Uses,”which is incorporated by reference, provides further discussion of PEGand forms thereof.

IX. Measurement of Potency, Functional In Vivo Half-Life, andPharmacokinetic Parameters

An important aspect of the invention is the prolonged biologicalhalf-life that is obtained by construction of the hGH polypeptide withor without conjugation of the polypeptide to a water soluble polymermoiety. The rapid decrease of hGH polypeptide serum concentrations hasmade it important to evaluate biological responses to treatment withconjugated and non-conjugated hGH polypeptide and variants thereof. Theconjugated and non-conjugated hGH polypeptide and variants thereof ofthe present invention may have prolonged serum half-lives also aftersubcutaneous or i.v. administration, making it possible to measure by,e.g. ELISA method or by a primary screening assay. ELISA or RIA kitsfrom either BioSource International (Camarillo, Calif.) or DiagnosticSystems Laboratories (Webster, Tex.) may be used. Measurement of in vivobiological half-life is carried out as described herein.

The potency and functional in vivo half-life of an hGH polypeptidecomprising a non-naturally encoded amino acid can be determinedaccording to the protocol described in Clark, R., et al., J. Biol. Chem.271(36):21969-21977 (1996).

Pharmacokinetic parameters for a hGH polypeptide comprising anon-naturally encoded amino acid can be evaluated in normalSprague-Dawley male rats (N=5 animals per treatment group). Animals willreceive either a single dose of 25 ug/rat iv or 50 ug/rat sc, andapproximately 5-7 blood samples will be taken according to a pre-definedtime course, generally covering about 6 hours for a hGH polypeptidecomprising a non-naturally encoded amino acid not conjugated to a watersoluble polymer and about 4 days for a hGH polypeptide comprising anon-naturally encoded amino acid and conjugated to a water solublepolymer. Pharmacokinetic data for hGH polypeptides is well-studied inseveral species and can be compared directly to the data obtained forhGH polypeptides comprising a non-naturally encoded amino acid. SeeMordenti J., et al., Pharm. Res. 8(11): 1351-59 (1991), which isincorporated by reference herein, for studies related to hGH.

Pharmacokinetic parameters can also be evaluated in a primate, e.g.,cynomolgus monkeys. A single injection may be administered eithersubcutaneously or intravenously, and serum hGH levels are monitored overtime.

The specific activity of hGH polypeptides in accordance with thisinvention can be determined by various assays known in the art. Thebiological activity of the hGH polypeptide muteins, or fragmentsthereof, obtained and purified in accordance with this invention can betested by methods described or referenced herein or known to those ofordinary skill in the art.

X. Administration and Pharmaceutical Compositions

The polypeptides or proteins of the invention (including but not limitedto, hGH, synthetases, proteins comprising one or more unnatural aminoacid, etc.) are optionally employed for therapeutic uses, including butnot limited to, in combination with a suitable pharmaceutical carrier.Such compositions, for example, comprise a therapeutically effectiveamount of the compound, and a pharmaceutically acceptable carrier orexcipient. Such a carrier or excipient includes, but is not limited to,saline, buffered saline, dextrose, water, glycerol, ethanol, and/orcombinations thereof. The formulation is made to suit the mode ofadministration. In general, methods of administering proteins are knownto those of ordinary skill in the art and can be applied toadministration of the polypeptides of the invention.

Therapeutic compositions comprising one or more polypeptide of theinvention are optionally tested in one or more appropriate in vitroand/or in vivo animal models of disease, to confirm efficacy, tissuemetabolism, and to estimate dosages, according to methods known to thoseof ordinary skill in the art. In particular, dosages can be initiallydetermined by activity, stability or other suitable measures ofunnatural herein to natural amino acid homologues (including but notlimited to, comparison of a hGH polypeptide modified to include one ormore unnatural amino acids to a natural amino acid hGH polypeptide),i.e., in a relevant assay.

Administration is by any of the routes normally used for introducing amolecule into ultimate contact with blood or tissue cells. The unnaturalamino acid polypeptides of the invention are administered in anysuitable manner, optionally with one or more pharmaceutically acceptablecarriers. Suitable methods of administering such polypeptides in thecontext of the present invention to a patient are available, and,although more than one route can be used to administer a particularcomposition, a particular route can often provide a more immediate andmore effective action or reaction than another route.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositions of thepresent invention.

hGH polypeptides of the invention, including but not limited toPEGylated hGH, may be administered by any conventional route suitablefor proteins or peptides, including, but not limited to parenterally,e.g. injections including, but not limited to, subcutaneously orintravenously or any other form of injections or infusions. Polypeptidecompositions can be administered by a number of routes including, butnot limited to oral, intravenous, intraperitoneal, intramuscular,transdermal, subcutaneous, topical, sublingual, or rectal means.Compositions comprising non-natural amino acid polypeptides, modified orunmodified, can also be administered via liposomes. Such administrationroutes and appropriate formulations are generally known to those ofskill in the art. The hGH polypeptide, including but not limited toPEGylated hGH, comprising a non-naturally encoded amino acid may be usedalone or in combination with other suitable components such as apharmaceutical carrier.

The hGH polypeptide comprising a non-natural amino acid, alone or incombination with other suitable components, can also be made intoaerosol formulations (i.e., they can be “nebulized”) to be administeredvia inhalation. Aerosol formulations can be placed into pressurizedacceptable propellants, such as dichlorodifluoromethane, propane,nitrogen, and the like.

Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, intraperitoneal, and subcutaneous routes, include aqueousand non-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations of hGH can be presented in unit-dose or multi-dosesealed containers, such as ampules and vials.

Parenteral administration and intravenous administration are preferredmethods of administration. In particular, the routes of administrationalready in use for natural amino acid homologue therapeutics (includingbut not limited to, those typically used for EPO, GH, G-CSF, GM-CSF,IFNs, interleukins, antibodies, and/or any other pharmaceuticallydelivered protein), along with formulations in current use, providepreferred routes of administration and formulation for the polypeptidesof the invention.

The viscosity and formulation profiles of the polypeptide compositionmay allow administration with a small gauge needle by using smallerplunger pressures compared to conventional formulations. Smaller gaugeneedles including, but not limited to, 27, 28, 29, 30, or 31 gaugeneedles may be used to administer the polypeptide composition of thepresent invention to subjects. U.S. Pat. No. 6,875,432, which isincorporated by reference herein, discusses the viscosity of proteinformulations and problems associated with viscosity of proteinformulations for subcutaneous administration. Small gauge needles are anadvantage due to reduced pain of injection which improves patientcompliance with dosing regimens. Smaller plunger pressures needed forinjecting render the hGH polypeptide, including but not limited toPEGylated hGH, easier to administer with short injection duration. Typesof needles include, but are not limited to, pen needles, thin walled,normal walled, and luer needles.

Viscosity may be measured by standard techniques known to those ofordinary skill in the art. “Viscosity” may be “kinematic viscosity” or“absolute viscosity.” “Kinematic viscosity” is a measure of theresistive flow of a fluid under the influence of gravity. When twofluids of equal volume are placed in identical capillary viscometers andallowed to flow by gravity, a viscous fluid takes longer than a lessviscous fluid to flow through the capillary. If one fluid takes 200seconds to complete its flow and another fluid takes 400 seconds, thesecond fluid is twice as viscous as the first on a kinematic viscosityscale. “Absolute viscosity”, sometimes called dynamic or simpleviscosity, is the product of kinematic viscosity and fluid density:

Absolute Viscosity=Kinematic Viscosity×Density

The dimension of kinematic viscosity is L²/T where L is a length and Tis a time. Commonly, kinematic viscosity is expressed in centistokes(cSt). The SI unit of kinematic viscosity is mm²/s, which is 1 cSt.Absolute viscosity is expressed in units of centipoise (cP). The SI unitof absolute viscosity is the milliPascal-second (mPa-s), where 1 cP=1mPa-s.

The dose administered to a patient, in the context of the presentinvention, is sufficient to have a beneficial therapeutic response inthe patient over time, or other appropriate activity, depending on theapplication. The dose is determined by the efficacy of the particularvector, or formulation, and the activity, stability or serum half-lifeof the unnatural amino acid polypeptide employed and the condition ofthe patient, as well as the body weight or surface area of the patientto be treated. The size of the dose is also determined by the existence,nature, and extent of any adverse side-effects that accompany theadministration of a particular vector, formulation, or the like in aparticular patient.

In determining the effective amount of the vector or formulation to beadministered in the treatment or prophylaxis of disease (including butnot limited to, cancers, inherited diseases, diabetes, AIDS, or thelike), the physician evaluates circulating plasma levels, formulationtoxicities, progression of the disease, and/or where relevant, theproduction of anti-unnatural amino acid polypeptide antibodies.

The dose administered, for example, to a 70 kilogram patient, istypically in the range equivalent to dosages of currently-usedtherapeutic proteins, adjusted for the altered activity or serumhalf-life of the relevant composition. The vectors or pharmaceuticalformulations of this invention can supplement treatment conditions byany known conventional therapy, including antibody administration,vaccine administration, administration of cytotoxic agents, naturalamino acid polypeptides, nucleic acids, nucleotide analogues, biologicresponse modifiers, and the like.

For administration, formulations of the present invention areadministered at a rate determined by the LD-50 or ED-50 of the relevantformulation, and/or observation of any side-effects of the unnaturalamino acid polypeptides at various concentrations, including but notlimited to, as applied to the mass and overall health of the patient.Administration can be accomplished via single or divided doses.

If a patient undergoing infusion of a formulation develops fevers,chills, or muscle aches, he/she receives the appropriate dose ofaspirin, ibuprofen, acetaminophen or other pain/fever controlling drug.Patients who experience reactions to the infusion such as fever, muscleaches, and chills are premedicated 30 minutes prior to the futureinfusions with either aspirin, acetaminophen, or, including but notlimited to, diphenhydramine. Meperidine is used for more severe chillsand muscle aches that do not quickly respond to antipyretics andantihistamines. Cell infusion is slowed or discontinued depending uponthe severity of the reaction.

Human hGH polypeptides of the invention can be administered directly toa mammalian subject. Administration is by any of the routes normallyused for introducing hGH polypeptide to a subject. The hGH polypeptidecompositions according to embodiments of the present invention includethose suitable for oral, rectal, topical, inhalation (including but notlimited to, via an aerosol), buccal (including but not limited to,sub-lingual), vaginal, parenteral (including but not limited to,subcutaneous, intramuscular, intradermal, intraarticular, intrapleural,intraperitoneal, inracerebral, intraarterial, or intravenous), topical(i.e., both skin and mucosal surfaces, including airway surfaces) andtransdermal administration, although the most suitable route in anygiven case will depend on the nature and severity of the condition beingtreated. Administration can be either local or systemic. Theformulations of compounds can be presented in unit-dose or multi-dosesealed containers, such as ampules and vials. hGH polypeptides of theinvention can be prepared in a mixture in a unit dosage injectable form(including but not limited to, solution, suspension, or emulsion) with apharmaceutically acceptable carrier or excipient. hGH polypeptides ofthe invention can also be administered by continuous infusion (using,including but not limited to, minipumps such as osmotic pumps), singlebolus or slow-release depot formulations.

Formulations suitable for administration include aqueous and non-aqueoussolutions, isotonic sterile solutions, which can contain antioxidants,buffers, bacteriostats, and solutes that render the formulationisotonic, and aqueous and non-aqueous sterile suspensions that caninclude suspending agents, solubilizers, thickening agents, stabilizers,and preservatives. Solutions and suspensions can be prepared fromsterile powders, granules, and tablets of the kind previously described.

Freeze-drying is a commonly employed technique for presenting proteinswhich serves to remove water from the protein preparation of interest.Freeze-drying, or lyophilization, is a process by which the material tobe dried is first frozen and then the ice or frozen solvent is removedby sublimation in a vacuum environment. An excipient may be included inpre-lyophilized formulations to enhance stability during thefreeze-drying process and/or to improve stability of the lyophilizedproduct upon storage. Pikal, M. Biopharm. 3(9)26-30 (1990) and Arakawaet al. Pharm. Res. 8(3):285-291 (1991).

The spray drying of pharmaceuticals is also known to those of ordinaryskill in the art. For example, see Broadhead, J. et al., “The SprayDrying of Pharmaceuticals,” in Drug Dev. Ind. Pharm, 18 (11 & 12),1169-1206 (1992). In addition to small molecule pharmaceuticals, avariety of biological materials have been spray dried and these include:enzymes, sera, plasma, micro-organisms and yeasts. Spray drying is auseful technique because it can convert a liquid pharmaceuticalpreparation into a fine, dustless or agglomerated powder in a one-stepprocess. The basic technique comprises the following four steps: a)atomization of the feed solution into a spray; b) spray-air contact; c)drying of the spray; and d) separation of the dried product from thedrying air. U.S. Pat. Nos. 6,235,710 and 6,001,800, which areincorporated by reference herein, describe the preparation ofrecombinant erythropoietin by spray drying.

The pharmaceutical compositions and formulations of the presentinvention may comprise a pharmaceutically acceptable carrier, excipient,or stabilizer. Pharmaceutically acceptable carriers are determined inpart by the particular composition being administered, as well as by theparticular method used to administer the composition. Accordingly, thereis a wide variety of suitable formulations of pharmaceuticalcompositions (including optional pharmaceutically acceptable carriers,excipients, or stabilizers) of the present invention (see, e.g.,Remington's Pharmaceutical Sciences, 17^(th) ed. 1985)).

Suitable carriers include, but are not limited to, buffers containingsuccinate, phosphate, borate, HEPES, citrate, histidine or histidinederivatives, imidazole, acetate, bicarbonate, and other organic acids;antioxidants including but not limited to, ascorbic acid; low molecularweight polypeptides including but not limited to those less than about10 residues; proteins, including but not limited to, serum albumin,gelatin, or immunoglobulins; hydrophilic polymers including but notlimited to, polyvinylpyrrolidone; amino acids including but not limitedto, glycine, glutamine, histidine or histidine derivatives, methionine,asparagine, arginine, glutamate, or lysine; monosaccharides,disaccharides, and other carbohydrates, including but not limited to,trehalose, sucrose, glucose, mannose, or dextrins; chelating agentsincluding but not limited to, EDTA; divalent metal ions including butnot limited to, zinc, cobalt, or copper; sugar alcohols including butnot limited to, mannitol or sorbitol; salt-forming counter ionsincluding but not limited to, sodium; and/or nonionic surfactantsincluding but not limited to Tween™ (including but not limited to, Tween80 (polysorbate 80) and Tween 20 (polysorbate 20; PS20)), Pluronics™ andother pluronic acids, including but not limited to, pluronic acid F68(poloxamer 188), or PEG. Suitable surfactants include for example butare not limited to polyethers based upon poly(ethyleneoxide)-poly(propylene oxide)-poly(ethylene oxide), i.e., (PEO-PPO-PEO),or poly(propylene oxide)-poly(ethylene oxide)-poly(propylene oxide),i.e., (PPO-PEO-PPO), or a combination thereof. PEO-PPO-PEO andPPO-PEO-PPO are commercially available under the trade names Pluronics™,R-Pluronics™, Tetronics™ and R-Tetronics™ (BASF Wyandotte Corp.,Wyandotte, Mich.) and are further described in U.S. Pat. No. 4,820,352incorporated herein in its entirety by reference. Otherethylene/polypropylene block polymers may be suitable surfactants. Asurfactant or a combination of surfactants may be used to stabilizePEGylated hGH against one or more stresses including but not limited tostress that results from agitation. Some of the above may be referred toas “bulking agents.” Some may also be referred to as “tonicitymodifiers.” Additional carriers include, but are not limited to,ammonium sulfate ((NH₄)₂SO₄). Ammonium sulfate ((NH₄)₂SO₄) may be usedin a formulation of the present invention at a range of about 0.1 mM toabout 200 mM, including but not limited to, 200 mM, 190 mM, 180 mM, 170mM, 160 mM, 150 mM, 140 mM, 130 mM, 120 mM, 110 mM, 100 mM, 95 mM, 90mM, 85 mM, 80 mM, 75 mM, 70 mM, 65 mM, 60 mM, 55 mM, 50 mM, 45 mM, 40mM, 35 mM, 30 mM, 25 mM, 20 mM, 15 mM, 10 mM, 5 mM, 1 mM, 0.9 mM, 0.8mM, 0.7 mM, 0.6 mM, 0.5 mM, 0.4 mM, 0.3 mM, 0.2 mM, and 0.1 mM.Histidine may be used in a formulation of the present invention at arange of about 0.1 mM to about 200 mM, including but not limited to, 200mM, 190 mM, 180 mM, 170 mM, 160 mM, 150 mM, 140 mM, 130 mM, 120 mM, 110mM, 100 mM, 95 mM, 90 mM, 85 mM, 80 mM, 75 mM, 70 mM, 65 mM, 60 mM, 55mM, 50 mM, 45 mM, 40 mM, 35 mM, 30 mM, 25 mM, 20 mM, 15 mM, 10 mM, 5 mM,1 mM, 0.9 mM, 0.8 mM, 0.7 mM, 0.6 mM, 0.5 mM, 0.4 mM, 0.3 mM, 0.2 mM,and 0.1 mM. In some embodiments, Histidine is between about 5 mM andabout 30 mM in the formulation.

A buffer of a formulation of the present invention may be in a range ofabout 0.1 mM to about 200 mM, including but not limited to, 200 mM, 190mM, 180 mM, 170 mM, 160 mM, 150 mM, 140 mM, 130 mM, 120 mM, 110 mM, 100mM, 95 mM, 90 mM, 85 mM, 80 mM, 75 mM, 70 mM, 65 mM, 60 mM, 55 mM, 50mM, 45 mM, 40 mM, 35 mM, 30 mM, 25 mM, 20 mM, 19 mM, 18 mM, 17 mM, 16mM, 15 mM, 14 mM, 13 mM, 12 mM, 11 mM, 10 mM, 9 mM, 8 mM, 7 mM, 6 mM, 5mM, 4 mM, 3 mM, 2 mM, 1 mM, 0.9 mM, 0.8 mM, 0.7 mM, 0.6 mM, 0.5 mM, 0.4mM, 0.3 mM, 0.2 mM, and 0.1 mM. In some embodiments, the buffer is at aconcentration of about 0.1 mM to about 200 mM. In some embodiments, thebuffer is at a concentration of about 1 mM to about 75 mM. In someembodiments, the buffer is at a concentration of about 1 mM to about 20mM. In some embodiments, the buffer is at a concentration of about 5 mMto about 30 mM. The buffer of a formulation of the present invention mayprovide a pH range from about pH 4.0 to about pH 8.5, including but notlimited to, pH 8.5, pH 8.0, pH 7.5, pH 7.0, pH 6.5, pH 6.0, pH 5.5, pH5.0, pH 4.5, and pH 4.0. The pH is any tenth pH value within thoseenumerated above; for example, pH 8.5, pH 8.4, pH 8.3, pH 8.2, pH 8.1,pH 8.0, pH 1.9, pH 7.8, pH 7.7, pH 7.6, pH 7.5, pH 7.4, pH 7.3, pH 7.2,pH 7.1, pH 7.0, pH 6.9, pH 6.8, pH 6.7, pH 6.6, pH 6.5, pH 6.4, pH 6.3,pH 6.2, pH 6.1, pH 6.0, pH 5.9, pH 5.8, pH 5.7, pH 5.6, pH 5.5, pH 5.4,pH 5.3, pH 5.2, pH 5.1, pH 5.0, pH 4.9, pH 4.8, pH 4.7, pH 4.6, pH 4.5,pH 4.4, pH 4.3, pH 4.2, pH 4.1, and pH 4.0. In some embodiments, the pHis between about pH 6.0 and about pH 7.3. In some embodiments, the pH isbetween about pH 5.5 and about 8.0. In some embodiments, the pH isbetween about 4.0 and about 8.5. In some embodiments, the pH is betweenabout 4.0 and about 7.5. In some embodiments, the pH is between about6.0 and about 7.5.

An amino acid of a formulation of the present invention may be in arange of about 0.1 g/L to 100 g/L, including but not limited to, 100g/L, 95 g/L, 90 g/L, 85 g/L, 80 g/L, 75 g/L, 70 g/L, 65 g/L, 60 g/L, 55g/L, 50 g/L, 45 g/L, 40 g/L, 35 g/L, 30 g/L, 25 g/L, 20 g/L, 19 g/L, 18g/L, 17 g/L, 16 g/L, 15 g/L, 14 g/L, 13 g/L, 12 g/L, 11 g/L, 10 g/L, 9g/L, 8 g/L, 7 g/L, 6 g/L, 5 g/L, 4 g/L, 3 g/L, 2 g/L, 1 g/L, 0.9 g/L,0.8 g/L, 0.7 g/L, 0.6 g/L, 0.5 g/L, 0.4 g/L, 0.3 g/L, 0.2 g/L, and 0.1g/L. In some embodiments, the amino acid is between about 0.1 g/L and 60g/L. In some embodiments, the amino acid is between about 0.1 g/L and100 g/L. In some embodiments, the amino acid is between about 1 g/L and50 g/L. In some embodiments, the amino acid is between about 5 g/L and25 g/L.

A sugar alcohol of a formulation of the present invention may be in arange of about 0.1 g/L to 100 g/L, including but not limited to, 100g/L, 95 g/L, 90 g/L, 85 g/L, 80 g/L, 75 g/L, 70 g/L, 65 g/L, 60 g/L, 55g/L, 50 g/L, 45 g/L, 40 g/L, 35 g/L, 30 g/L, 25 g/L, 20 g/L, 19 g/L, 18g/L, 17 g/L, 16 g/L, 15 g/L, 14 g/L, 13 g/L, 12 g/L, 11 g/L, 10 g/L, 9g/L, 8 g/L, 7 g/L, 6 g/L, 5 g/L, 4 g/L, 3 g/L, 2 g/L, 1 g/L, 0.9 g/L,0.8 g/L, 0.7 g/L, 0.6 g/L, 0.5 g/L, 0.4 g/L, 0.3 g/L, 0.2 g/L, and 0.1g/L. In some embodiments, the sugar alcohol is between about 0.1 g/L andabout 60 g/L. In some embodiments, the sugar alcohol is between about0.1 g/L and about 100 g/L. In some embodiments, the sugar alcohol isbetween about 1 g/L and about 50 g/L. In some embodiments, the sugaralcohol is between about 2 g/L and about 25 g/L.

A carbohydrate of a formulation of the present invention may be in arange of about 0.1 g/L to 100 g/L, including but not limited to, 100g/L, 95 g/L, 90 g/L, 85 g/L, 80 g/L, 75 g/L, 70 g/L, 65 g/L, 60 g/L, 55g/L, 50 g/L, 45 g/L, 40 g/L, 35 g/L, 30 g/L, 25 g/L, 20 g/L, 19 g/L, 18g/L, 17 g/L, 16 g/L, 15 g/L, 14 g/L, 13 g/L, 12 g/L, 11 g/L, 10 g/L, 9g/L, 8 g/L, 7 g/L, 6 g/L, 5 g/L, 4 g/L, 3 g/L, 2 g/L, 1 g/L, 0.9 g/L,0.8 g/L, 0.7 g/L, 0.6 g/L, 0.5 g/L, 0.4 g/L, 0.3 g/L, 0.2 g/L, and 0.1g/L. In some embodiments, the carbohydrate is at about 0.1 g/L to about100 g/L. In some embodiments, the carbohydrate is at about 1 g/L toabout 50 g/L. In some embodiments, the carbohydrate is at about 2 g/L toabout 25 g/L. In some embodiments, the carbohydrate is at about 0.1 g/Lto about 50 g/L.

A disaccharide of a formulation of the present invention may be in arange of about 0.1 g/L to 100 g/L, including but not limited to, 100g/L, 95 g/L, 90 g/L, 85 g/L, 80 g/L, 75 g/L, 70 g/L, 65 g/L, 60 g/L, 55g/L, 50 g/L, 45 g/L, 40 g/L, 35 g/L, 30 g/L, 25 g/L, 20 g/L, 19 g/L, 18g/L, 17 g/L, 16 g/L, 15 g/L, 14 g/L, 13 g/L, 12 g/L, 11 g/L, 10 g/L, 9g/L, 8 g/L, 7 g/L, 6 g/L, 5 g/L, 4 g/L, 3 g/L, 2 g/L, 1 g/L, 0.9 g/L,0.8 g/L, 0.7 g/L, 0.6 g/L, 0.5 g/L, 0.4 g/L, 0.3 g/L, 0.2 g/L, and 0.1g/L. In some embodiments, the disaccharide is between about 0.1 g/L andabout 50 g/L.

A monosaccharide of a formulation of the present invention may be in arange of about 0.1 g/L to 100 g/L, including but not limited to, 100g/L, 95 g/L, 90 g/L, 85 g/L, 80 g/L, 75 g/L, 70 g/L, 65 g/L, 60 g/L, 55g/L, 50 g/L, 45 g/L, 40 g/L, 35 g/L, 30 g/L, 25 g/L, 20 g/L, 19 g/L, 18g/L, 17 g/L, 16 g/L, 15 g/L, 14 g/L, 13 g/L, 12 g/L, 11 g/L, 10 g/L, 9g/L, 8 g/L, 7 g/L, 6 g/L, 5 g/L, 4 g/L, 3 g/L, 2 g/L, 1 g/L, 0.9 g/L,0.8 g/L, 0.7 g/L, 0.6 g/L, 0.5 g/L, 0.4 g/L, 0.3 g/L, 0.2 g/L, and 0.1g/L.

A non-ionic surfactant of a formulation of the present invention may bein a range of about 0.01% to about 10%, including but not limited to,10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%,0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%,0.02%, 0.01%, 0.009%, 0.0085%, 0.008%, 0.0075%, 0.007%, 0.0065%, 0.006%,0.0055%, 0.005%, 0.0045%, 0.004%, 0.0035%, 0.003%, 0.0025%, 0.002%,0.0015%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%,0.0003%, 0.0002%, and 0.0001%. In some embodiments, the non-ionicsurfactant is at about 0.0001% to about 10%. In some embodiments, thenon-ionic surfactant is at about 0.01% to about 10%. In someembodiments, the non-ionic surfactant is at about 0.1% to about 5%. Insome embodiments, the non-ionic surfactant is at about 0.1% to about 1%.In some embodiments, the non-ionic surfactant is at about 0.0001% toabout 1%.

The pharmaceutical quantity of hGH in a formulation of the presentinvention may be in a range of about 0.1 mg to about 50 mg, includingbut not limited to, 50 mg, 49 mg, 48 mg, 47 mg, 46 mg, 45 mg, 44 mg, 43mg, 42 mg, 41 mg, 40 mg, 39 mg, 38 mg, 37 mg, 36 mg, 35 mg, 34 mg, 33mg, 32 mg, 31 mg, 30 mg, 29.5 mg, 29 mg, 28.5 mg, 28 mg, 27.5 mg, 27 mg,26.5 mg, 26 mg, 25.5 mg, 25 mg, 24.5 mg, 24 mg, 23.5 mg, 23 mg, 22.5 mg,22 mg, 21.5 mg, 21 mg, 20.5 mg, 20 mg, 19.5 mg, 19 mg, 18.5 mg, 18 mg,17.5 mg, 17 mg, 16.5 mg, 16 mg, 15.5 mg, 15 mg, 14.5 mg, 14 mg, 13.5 mg,13 mg, 12.5 mg, 12 mg, 11.5 mg, 11 mg, 10.5 mg, 10 mg, 9.5 mg, 9 mg, 8.5mg, 8 mg, 7.5 mg, 7.0 mg, 6.5 mg, 6.0 mg, 5.5 mg, 5.0 mg, 4.5 mg, 4.0mg, 3.5 mg, 3.0 mg, 2.5 mg, 2.0 mg, 1.5 mg, 1.0 mg, 0.9 mg, 0.8 mg, 0.7mg, 0.6 mg, 0.5 mg, 0.4 mg, 0.3 mg, 0.2 mg, and 0.1 mg. In someembodiments, the pharmaceutical quantity of hGH in a formulation isbetween about 2 mg and about 30 mg. In some embodiments, thepharmaceutical quantity of hGH in a formulation is between about 2 mgand about 25 mg. In some embodiments, the pharmaceutical quantity of hGHin a formulation is between about 0.1 mg and about 30 mg. In someembodiments, the pharmaceutical quantity of hGH in a formulation isbetween about 0.1 mg and about 8 mg. In some embodiments, thepharmaceutical quantity of hGH in a formulation is between about 0.5 mgand about 8 mg. In some embodiments, the pharmaceutical quantity of hGHin a formulation is between about 0.5 mg and about 6 mg. In someembodiments, the pharmaceutical quantity of hGH in a formulation isbetween about 1 mg and about 5 mg.

hGH polypeptides of the invention, including those linked to watersoluble polymers such as PEG can also be administered by or as part ofsustained-release systems. Sustained-release compositions include,including but not limited to, semi-permeable polymer matrices in theform of shaped articles, including but not limited to, films, ormicrocapsules. Sustained-release matrices include from biocompatiblematerials such as poly(2-hydroxyethyl methacrylate) (Langer et al., J.Biomed. Mater. Res., 15: 267-277 (1981); Langer, Chem. Tech., 12: 98-105(1982), ethylene vinyl acetate (Langer et al., supra) orpoly-D-(−)-3-hydroxybutyric acid (EP 133,988), polylactides (polylacticacid) (U.S. Pat. No. 3,773,919; EP 58,481), polyglycolide (polymer ofglycolic acid), polylactide co-glycolide (copolymers of lactic acid andglycolic acid) polyanhydrides, copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (Sidman et al., Biopolymers, 22, 547-556 (1983),poly(ortho)esters, polypeptides, hyaluronic acid, collagen, chondroitinsulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides,nucleic acids, polyamino acids, amino acids such as phenylalanine,tyrosine, isoleucine, polynucleotides, polyvinyl propylene,polyvinylpyrrolidone and silicone. Sustained-release compositions alsoinclude a liposomally entrapped compound. Liposomes containing thecompound are prepared by methods known per se: DE 3,218,121; Eppstein etal., Proc. Natl. Acad. Sci. U.S.A., 82: 3688-3692 (1985); Hwang et al.,Proc. Natl. Acad. Sci. U.S.A., 77: 4030-4034 (1980); EP 52,322; EP36,676; U.S. Pat. No. 4,619,794; EP 143,949; U.S. Pat. No. 5,021,234;Japanese Pat. Appln. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545;and EP 102,324. All references and patents cited are incorporated byreference herein.

Liposomally entrapped hGH polypeptides can be prepared by methodsdescribed in, e.g., DE 3,218,121; Eppstein et al., Proc. Natl. Acad.Sci. U.S.A., 82: 3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci.U.S.A., 77: 4030-4034 (1980); EP 52,322; EP 36,676; U.S. Pat. No.4,619,794; EP 143,949; U.S. Pat. No. 5,021,234; Japanese Pat. Appln.83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324.Composition and size of liposomes are well known or able to be readilydetermined empirically by one of ordinary skill in the art. Someexamples of liposomes as described in, e.g., Park J W, et al., Proc.Natl. Acad. Sci. USA 92:1327-1331 (1995); Lasic D and Papahadjopoulos D(eds): MEDICAL APPLICATIONS OF LIPOSOMES (1998); Drummond D C, et al.,Liposomal drug delivery systems for cancer therapy, in Teicher B (ed):CANCER DRUG DISCOVERY AND DEVELOPMENT (2002); Park J W, et al., Clin.Cancer Res. 8:1172-1181 (2002); Nielsen U B, et al., Biochim. Biophys.Acta 1591(1-3):109-118 (2002); Mamot C, et al., Cancer Res. 63:3154-3161 (2003). All references and patents cited are incorporated byreference herein.

The dose administered to a patient in the context of the presentinvention should be sufficient to cause a beneficial response in thesubject over time. Generally, the total pharmaceutically effectiveamount of the hGH polypeptide of the present invention administeredparenterally per dose is in the range of about 0.01 μg/kg/day to about100 μg/kg, or about 0.05 mg/kg to about 1 mg/kg, of patient body weight,although this is subject to therapeutic discretion. The frequency ofdosing is also subject to therapeutic discretion, and may be morefrequent or less frequent than the commercially available hGHpolypeptide products approved for use in humans. Generally, a PEGylatedhGH polypeptide of the invention can be administered by any of theroutes of administration described above.

XI. Therapeutic Uses of hGH Polypeptides of the Invention

The hGH polypeptides of the invention are useful for treating a widerange of disorders.

The hGH agonist polypeptides of the invention may be useful, forexample, for treating growth deficiency, immune disorders, and forstimulating heart function. Individuals with growth deficienciesinclude, e.g., individuals with Turner's Syndrome, GH-deficientindividuals (including children), children who experience a slowing orretardation in their normal growth curve about 2-3 years before theirgrowth plate closes (sometimes known as “short normal children”), andindividuals where the insulin-like growth factor-I (IGF-I) response toGH has been blocked chemically (i.e., by glucocorticoid treatment) or bya natural condition such as in adult patients where the IGF-I responseto GH is naturally reduced. The hGH polypeptides of the invention may beuseful for treating individuals with the following conditions: pediatricgrowth hormone deficiency, idiopathic short stature, adult growthhormone deficiency of childhood onset, adult growth hormone deficiencyof adult onset, or secondary growth hormone deficiency. Adults diagnosedwith growth hormone deficiency in adulthood may have had a pituitarytumor or radiation. Conditions including but not limited to, metabolicsyndrome, head injury, obesity, osteoporosis, or depression may resultin growth hormone deficiency-like symptoms in adults.

An agonist hGH variant may act to stimulate the immune system of amammal by increasing its immune function, whether the increase is due toantibody mediation or cell mediation, and whether the immune system isendogenous to the host treated with the hGH polypeptide or istransplanted from a donor to the host recipient given the hGHpolypeptide (as in bone marrow transplants). “Immune disorders” includeany condition in which the immune system of an individual has a reducedantibody or cellular response to antigens than normal, including thoseindividuals with small spleens with reduced immunity due to drug (e.g.,chemotherapeutic) treatments. Examples individuals with immune disordersinclude, e.g., elderly patients, individuals undergoing chemotherapy orradiation therapy, individuals recovering from a major illness, or aboutto undergo surgery, individuals with AIDS, Patients with congenital andacquired B-cell deficiencies such as hypogammaglobulinemia, commonvaried agammaglobulinemia, and selective immunoglobulin deficiencies(e.g., IgA deficiency, patients infected with a virus such as rabieswith an incubation time shorter than the immune response of the patient;and individuals with hereditary disorders such as diGeorge syndrome.

hGH antagonist polypeptides of the invention may be useful for thetreatment of gigantism and acromegaly, diabetes and complications(diabetic retinopathy, diabetic neuropathy) arising from diabetes,vascular eye diseases (e.g., involving proliferativeneovascularization), nephropathy, and GH-responsive malignancies.

Vascular eye diseases include, e.g., retinopathy (caused by, e.g.,prematurity or sickle cell anemia) and macular degeneration.

GH-responsive malignancies include, e.g., Wilm's tumor, sarcomas (e.g.,osteogenic sarcoma), breast, colon, prostate, and thyroid cancer, andcancers of tissues that express GH receptor mRNA (i.e., placenta,thymus, brain, salivary gland, prostate, bone marrow, skeletal muscle,trachea, spinal cord, retina, lymph node and from Burkitt's lymphoma,colorectal carcinoma, lung carcinoma, lymphoblastic leukemia, andmelanoma).

The GH, e.g., hGH agonist polypeptides of the invention may be useful,for example, for treating chronic renal failure, growth failureassociated with chronic renal insufficiency (CRI), short statureassociated with Turner Syndrome, pediatric Prader-Willi Syndrome (PWS),HIV patients with wasting or cachexia, children born small forgestational age (SGA), obesity, and osteoporosis.

Average quantities of the hGH may vary and in particular should be basedupon the recommendations and prescription of a qualified physician. Theexact amount of hGH is a matter of preference subject to such factors asthe exact type of condition being treated, the condition of the patientbeing treated, as well as the other ingredients in the composition. Theamount to be given may be readily determined by one of ordinary skill inthe art based upon therapy with hGH.

Pharmaceutical compositions of the invention may be manufactured inconventional manner.

XII. Articles of Manufacture

In another embodiment of the invention, an article of manufacture isprovided which contains the formulation of the present invention andprovides instructions for its use. The article of manufacture comprisesa container. The article of manufacture may contain the formulation ofthe present invention, including but not limited to, lyophilized,liquid, spray dried, frozen or other forms thereof, and instructions forits preparation or reconstitution, if required. Suitable containersinclude, but are not limited to, for example, bottles, vials, syringes,auto-injection devices, and test tubes. The container may be formed froma variety of materials such as glass or plastic. The container holds theformulation and the label on, or associated with, the container mayindicate directions for reconstitution of the lyophilized formulation,if required, and/or use. For example, the label may indicate that theformulation is reconstituted to specific protein concentrations. Thelabel may further indicate that the formulation is useful or intendedfor subcutaneous administration. The container holding the formulationmay be a single-use or a multi-use vial. In one embodiment, thecontainer holding the formulation is a single-use vial. The article ofmanufacture may further comprise a second container comprising asuitable diluent. In one embodiment, the suitable diluent is sterilewater or bacteriostatic water for injection, USP. Upon mixing of adiluent and the lyophilized formulation, if required, the final proteinconcentration in the formulation may generally be between about 2 mg/mland about 50 mg/ml. Upon mixing of a diluent and the lyophilizedformulation, if required, the final protein concentration in theformulation may generally be between about 2 mg/ml and about 25 mg/ml.The final protein concentration in the formulation may generally bebetween about 2 mg/ml and about 25 mg/ml. In one embodiment, the finalprotein concentration is about 8 mg/ml. The article of manufacture mayfurther include other materials desirable from a commercial and userstandpoint, including other buffers, diluents, filters, needles,syringes, and package inserts with instructions for use.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Materials and Methods for Formulations for Met-Y35pAF hGH

This example describes a formulations study that identified andevaluated suitable conditions and excipients that preserve the proteinstructure and activity of Met-Y35pAF hGH during storage beforeconjugation with PEG. Liquid formulations of hGH are frozen formulationswhich contain (1) 2.5 g/L sodium bicarbonate, 20 g/L glycine, 2 g/Lmannitol, 2 g/L lactose, at pH 7.3; or 2) sodium citrate, 20 g/Lglycine, 5 g/L mannitol, at pH 6.0. An estimated dose of 0.5-4 mg/ml ofPEG-hGH is added as an active ingredient. These formulations arecurrently stored at −20° C. Development of a single-dose lyophilizedformulation of PEGylated hGH comprising a non-naturally encoded aminoacid or liquid formulation of PEGylated hGH comprising a non-naturallyencoded amino acid is desired.

Methods

Several methods were implemented to characterize the physical andchemical stability of Met-Y35pAF hGH. These methods may be used in theformulations study with PEGylated hGH.

SDS-PAGE with a Reducing Gel (SDS-PAGE-R) provided separations of hGHbased exclusively on molecular weight allowing any potential degradationproducts to be visualized, since this technique results in all disulfidebonds being completely cleaved and causes the polypeptides to becompletely unfolded. SDS-PAGE with a Non-reducing Gel (SDS-PAGE-NR)provided separation of molecules based on molecular weight, allowing theidentification of potential dimers of hGH since the protein is unfoldedwithout a reducing agent, thus leaving disulfide bonds intact.

Differential Scanning Calorimetry (DSC) was used to measure the ΔH(enthalpy) of unfolding due to heat denaturation. This techniqueevaluated the effects of different solution conditions and excipients onprotein stability which was reflected in either an increase or decreasein the overall melting temperature (T_(m)) of the protein. Anothertechnique used was Reverse Phase HPLC (RP-HPLC) to separate molecules onthe basis of relative hydrophobicities. Specifically, this method wasused to separate hGH based on subtle differences in hydrophobicity andretention behavior associated with structural modifications such asdeamidation and oxidation.

Matrix and Solutions

A Formulations Matrix of forty-eight combinations was designed witheither U.S.P. (U.S. Pharmacopeia) grade or high analytical gradeexcipients. The matrix, sample preparation, and procedures used were asfollows (Table 1):

A B C D E F 20 mM 20 mM 50 mM 50 mM 50 mM 50 mM Sodium Acetate SodiumCitrate Histidine Sodium Phosphate Sodium Bicarbonate HEPES Excipients*pH 4.5 pH 6.0 pH 6.5 pH 7.0 pH 7.3 pH 7.5 1 No additions 2 15 mMTrehalose 3 15 mM Sucrose 4 15 mM Mannitol 5 75 mM (NH₄)₂SO₄ 6 250 mM Glycine 7 250 mM  Arginine 8 100 mM  Glutamate

Stocks of all excipients were sterile filtered, dated and stored at 4°C. Sugars were made as 100 mM stocks, (NH₄)SO₄ as 1 M stock, and aminoacids at the following stock concentrations: 1M Glycine, 666 mMArginine, or 1M Glutamate.

The specific buffers were prepared the day of experiment as 250 mM to 1Mstocks (sterile filtered and dated), and excipients were added to thefinal concentration, sterile filtered and stored at 4° C.

Sample Preparation

The sample of methionyl-hGH with the non-natural amino acidp-acetyl-phenylalanine substituted at position 35 (MetY35pAF cB1) in WHObuffer was allocated into 6 different vials at 4.425 mg/ml and frozen at−80° C. until the day of the study. On the day of experiment, MetY35pAFwas buffer exchanged into the specific buffer at 4° C. by dialysis in0.1-0.5 mL Slide-a-lyzers. Approximately 800 μl of 425 mg/ml Met-Y35pAFhGH was dialyzed in 250 mL buffer for at least four hours, and thedialysis buffer was changed (250 mL) for overnight dialysis. Using 500mL provided a 10,000,000-fold dilution of WHO buffer excipients.

Samples were removed from dialysis and concentrations were measured.Each sample was diluted to the appropriate concentration. For DSC, thedesired final concentration was 0.75 mg/ml (450 ul volume). For RP-HPLC,the final concentration was 2 mg/ml (30 ul volume). Ten samples wereanalyzed on RP-HPLC for each individual group of the matrix. Enoughsample to allow for four time points (3 days, 1 week, 2 weeks, and 1months) was stored in a tube at 4° C. Enough sample for 5 freeze/thawcycles was stored in a vial at −80° C. There may have been one or twoadditional freeze/thaw cycles prior to the number of freeze/thaw cycles(1, 2, 3, 4 and 5 cycles) completed as part of this study. The samplefor the 0 time point at 4° C. was immediately placed in an HPLC vial.

For SDS-PAGE-R, 30 ug (0.015 mL@2 mg/mL) of material was used in theanalysis. Samples were aliquoted into three vials as indicated forRP-HPLC.

For SDS-PAGE-NR, 30 ug (0.015 mL@ 2 mg/mL) of material was used in theanalysis. Samples were aliquoted into three vials as indicated forRP-HPLC. In all cases, any remaining sample was immediately frozen at−80° C.

Example 2 Results of Formulation Study for Met-Y35pAF hGH Buffer pH

The pH of all formulation buffers was analyzed after six weeks at 4° C.It was found that the pH of all buffers chosen was stable over a sixweek period, with the exception of the sodium bicarbonate buffer (GroupE) as shown in FIG. 1. The pH of Group E increased over time.

SDS-PAGE-R and SDS-PAGE-NR

Analysis of samples with SDS-PAGE reducing and non-reducing gels showedno change either after four weeks (1 month) at 4° C. or during fivefreeze/thaw cycles. No dimer formation or degradation products werefound by this method. Reducing gels for group A (at t=0, t=4 weeks at 4°C.) are shown as FIGS. 2C and 2D, and non-reducing gels for group B (att=0, t=4 weeks at 4° C.) are shown as FIGS. 2A and 2B.

Differential Scanning Calorimetry (DSC)

The effects of buffer and excipient on thermal stability of MetY35pAFhGH are shown as FIGS. 3-8. FIG. 3A-F shows overlaid thermoprofiles offormulation groups A-F. FIG. 5 provides a table summarizing the DSCmelting temperatures and changes to T_(m) for the full matrix.Thermoprofiles from B7 and F2 of the matrix are shown in FIG. 4A-B.

Buffer A demonstrated the lowest T_(m), as shown in FIGS. 5 and 6.Arginine raised the T_(m) in Groups B and C, as shown in B7 and C7 ofthe matrix on FIGS. 5 and 6. At higher pHs (i.e. pH>7.0), MetY35pAF hGHexhibited greater thermostability; however additional subpopulationswere seen that have much higher melting temperatures and broader curvesas shown in FIGS. 3 and 4. These subpopulations may exhibit greaterstability or aggregation tendencies. Amino acids, in particular Arg andGlu, as well as (NH₄)₂SO₄ decreased the T_(m) at higher pHs. Sugars didnot have an effect on the overall T_(m).

The ratio of ΔH_(v)/ΔH was analyzed for all groups as shown in FIG. 7.Recombinant hGH is known to exhibit a ΔH_(v)/ΔH>4. Variations in thisratio were observed; these values demonstrate that the unfolding ofMetY35pAF is irreversible. As the pH increased, additionalsubpopulations were identified (the ratio for a 2nd subpopulation ofGroup F was also plotted at pH 7.5 on FIG. 7). The change in enthalpy(AUC) of each sample was similar as shown in FIG. 8, except at higherpHs. Differences in ΔH at higher pHs may be due to missed broadtransitions, accounting for a lower AUC. ΔH (AUC of the fit) was plottedfor every group to ensure similar values were obtained within eachgroup.

Reverse Phase HPLC (RP-HPLC)

RP-HPLC was used to analyze the purity and chemical degradation ofMetY35pAF hGH during storage at 4° C. for four weeks. Datasets forGroups B, C, E and F each comparing MetY35pAF hGH with the WHO hGHstandard are shown in FIGS. 9A-D. FIG. 10A illustrates the use ofAgilent Chemstation software in analyzing the different peaks present ingroup E5 (the main peak, primary deamidation/oxidation peak, and thesecondary deamidation peak). The primary deamidation/oxidation peakincreased over time with Group E as shown in FIGS. 10B and 11; also,higher pHs exhibited greater amounts of deamidation/oxidation than lowerpHs. FIG. 12 shows that the secondary deamidation/oxidation peakdemonstrated less variability over time with all formulations groups.The main GH peak decreased over time for all formulations groups asshown in FIG. 13, though groups B and C exhibited less change comparedto the other groups. Group A exhibited very little deamidation, but themain GH peak did decrease over time and additional peaks, other thanknown deamidation/oxidation peaks, did develop over time. The data ofGroup A did, however, demonstrate that MetY35pAF is fairly stable from 0to 3 days at this pH, which is critical since the PEGylation reactiontakes place at pH 4.0 over one to two days.

RP-HPLC was also used to analyze the samples that had undergonefreeze-thaw cycles. Individual groups within B and C appeared similar asshown in FIG. 14 (primary deamidation/oxidation peak) and FIG. 15(secondary deamidation peak). After two freeze/thaw cycles, the main GHpeak decreased as shown in FIG. 16, indicating freeze/thaw effects.However, one or two freeze/thaw cycles did occur with this sample priorto the initiation of this formulations study.

Summary of Findings

It is well established that hGH exhibits a highly flexible structure atlower pH, and hGH forms a very rigid structure at higher pH (Kasimova etal. J. Mol. Biol. 2002 318:679-695).

Of the buffers tested, it was found that Buffer B (20 mM sodium citrate)at pH 6.0 performed best for chemical and thermodynamic stability ofMetY35pAF hGH. The addition of Arginine at pH 6.0 increased thethermostability of MetY35pAF hGH. At this time, sugars did not have anypositive or negative effect on MetY35pAF hGH; however, sugars may beimportant for pH shifts pre- and post-PEGylation events. The oximelinkage over time is also likely to be more stable at pH 6.0 than at ahigher pH such as pH 7.5.

Example 3

The stability profile of PEG-hGH is examined with key formulationparameters under accelerated stability conditions, major degradationproducts are identified, and stability indicating assays are confirmed.The main objective of single-dose formulation development is to optimizethe formulation for sufficient storage stability minimally atrefrigerated temperature and may have sufficient storage stability whenstored at ambient temperature. In addition to the lyophilizedformulation development, additional studies are investigating thesensitivity to light exposure and agitation, development of the RP-HPLCmethod distinguishing the PEGylated hGH from the non-PEGylated form,structural analysis, and other studies. Successful lyophilizedformulations normally show the following attributes: stability insolution for handling during formulation and fill-finish, good stabilityduring freeze-drying, good storage stability, native structure in thedried state (if relevant), no obvious sample collapse or melt back,optimum moisture content level, high glass transition temperature,rugged cake structure, efficient drying cycle, minimum injectionassociated pain, and dissolves in water-filled injectable within oneminute when reconstituted. In some embodiments, the optimum moisturecontent level is <3% water for a lyophilized formulation. In preferredembodiments, the optimum moisture content level is <1% water for alyophilized formulation.

The formulation variables are determined. The stability of lyophilizedformulation candidates are examined at 40° C., ambient temperature, andrefrigerated temperature with weekly time points for up to 4 weeks, 6weeks, and 2 months. Also, the stability of lyophilized formulationcandidates are examined at ambient temperature and refrigeratedtemperature for up to 3, 4, and 6 month increments. The stability of thereconstituted form of each formulation during storage at 2-8° C. for aweek is also be tested. Analytical methods include the SDS-PAGE methoddescribed above, SEC-HPLC, Ion-exchange HPLC, RP-HPLC, and otherstructural analyses.

Basic parameters useful for optimizing lyophilization cycle for moststable formulations are determined. For example, collapse temperature(or glass transition temperature), annealing temperature, and otherimportant physical properties of frozen formulations are determined. Atest lyophilization process is run to confirm that ideal cake can beobtained with the optimized cycle.

Example 4 Reverse Phase High Performance Liquid Chromatography (RP-HPLC)

Reverse Phase High Performance Liquid Chromatography (RP-HPLC) is atechnique that separates molecules on the basis of relativehydrophobicities. Samples are passed over a stationary phase of silicacovalently bonded to hydrocarbon chains. The molecules of interest areretarded by the stationary phase and eluted with an isocratic solvent.The chromatographic elution time is characteristic for a particularmolecule. This method separates hGH based on subtle differences inhydrophobicity and retention behavior associated with structuralmodifications such as deamidation.

C4 RP-HPLC was used to assess relative purity and potential chemicaldegradation (deamidation and oxidation) of recombinant human growthhormone (hGH). This method was used to support identification and purityassessment of hGH. Some partial degradation products of hGH wereobserved using this technique. References for this technique include,European Pharmacopoeia 2002, p. 193; British Pharmacopoeia 2001, p.1938-1939; and “A Reversed-Phase High Performance Liquid ChromatographicMethod for Characterization of Biosynthetic Human Growth Hormone” by R.M. Riggin et al. Analytical Biochemistry 167, 199-209 (1987).

Equipment for this procedure included, the following or equivalentsthereof: UV/Vis Spectrophotometer (Agilent 8453 or equivalent); 50 ulquartz cuvette; PD-10, Nap-10, or Nap 5 desalting columns (depending onsample volume; Amersham Biosciences Nap5 column 17-0853-02 orequivalent); 0.5 mL Vivaspin concentrators (if needed; Vivascience10,000 MWCO, PES, VS0102 or equivalent); HPLC vials and caps (Alltech100 ul screw cap polypropylene vials #12962, TFE liner caps #73048, openhole screw caps #73044, or equivalent); clean 1 and 2 L glass bottles; acolumn such as Vydac C4 214TP54, a C4-silica reversed phase HPLC columnwith a dimension of 4.6×250 mm, particle size of 5μ and pore size of 300Å; and High-pressure liquid chromatography instrument capable ofperforming linear gradients (such as Agilent 1100 HPLC equipped with avacuum degasser, quaternary pump, thermostatted autosampler,thermostatted column compartment, diode array detector (DAD), andChemstation chromatography software).

Reagents for this procedure included solid chemicals that wereanalytical grade or better and solvents that were HPLC grade or better,unless otherwise noted. Examples of such chemicals includeTRIS-Tromethamine, U.S.P. grade, Spectrum TR149, or equivalent;N-propanol, HPLC grade, 99.9%, Sigma Aldrich 34871, or equivalent; andAmmonium Bicarbonate, Ultra >99.5%, Fluka #09830, or equivalent.Additional solutions include: Buffer for Deamidation Control (30 mMAmmonium Bicarbonate, pH 9.0) and mobile phase solution. For the bufferfor deamidation control, 2.37 g of Ammonium bicarbonate was dissolved in0.95 L Milli-Q H₂O, pH to 9.0 with NaOH, and the volume was brought upto 1 L with Milli-Q H₂O. The resulting buffer was sterile filtered using0.22 μm PES filters (Corning #431098, or equivalent). For the Mobilephase solution (50 mM Tris-HCl, pH 7.5 and 29% n-propanol)), 6.05 gTromethamine (USP grade, Spectrum #TR149, or equivalent) was dissolvedin 0.95 L Milli-Q H₂O. The solution was brought to pH 7.5 with HCl, andthe volume brought up to 1 L with Milli-Q H₂O. The two solvents (TRISand propanol) were mixed, and the mixture was sterile filtered using0.22 μm PES filters (Corning #431098, or equivalent).

Samples for use as standards in RP-HPLC include World Health,Organization (WHO) hGH (Cat. #98/574) reconstituted to 1.9-2.1 mg/mlwith 1.0 ml of water. Other hGH reference standards may be used at1.9-2.1 mg/ml concentration. For the resolution solution, the hGHstandard is buffer exchanged into 30 mM Ammonium Bicarbonate, pH 9.0buffer using a PD-10, Nap-10, or Nap-5 desalting column (depending onsample volume). The standard was concentrated using a 0.5 mL Vivaspinconcentrator to 1.9-2.1 mg/ml, incubated at 37° C. for 24 hours, andthen stored at −80° C. until use. Test material was diluted to 2.0 mg/mlprotein concentration for analysis. Sample concentrations were measuredusing standard techniques.

Procedure

The instrument was set-up with the following conditions: 1) Column:Vydac C4 214TP54 column; 2) Pump Setup—gradient: isocratic; flow rate:0.5 ml/min; duration: 90 min; Max Pressure: 200 bar; 3) AutosamplerTemperature: 4° C.; 4) Injector Setup—Injection: Standard Injection;Injection Volume: 20 μl; Draw Speed: 100 μl/min; Needle Wash: 100 μlwith water; Injection Speed: 100 μl/min; Stop Time: As pump; 5) DADSignals—Table 2; Peak Width >0.1 min; Slit: 4 nm; Stop Time: 75 min;

TABLE 2 Sample Bw Reference Bw Units 220 4 600 100 nm 276 4 600 100 nm214 8 600 100 nm 220 4 600 100 nm

6) Column Thermostat—Temperature: 45° C.; Store: Temperature; 7)Preliminary Integration Events-Slope Sensitivity: 0.1; Peak Width: 0.5;Area Reject: 1.0; Height Reject: 1.0; Integration ON: 10 min.

The column was equilibrated with 10 column volumes (41.5 ml=83 min at0.5 ml/min) of the mobile phase. 20 μl of the standard was injectedusing the autosampler, and the HPLC program was run. If the retentiontime of the WHO standard was not between 33-35 minutes, or the otherstandard was not between 37-40 minutes, the mobile phase composition wasadjusted, the column was re-equilibrated, and the standard was re-run.Suggested adjustments included adding less than 5 ml of 50 mM Tris-HClpH 7.5 per liter of mobile phase if retention time was less than 33minutes, and less than 2 ml of n-propanol if retention time greater than35. Since evaporation of the propanol may occur, a standard was run oneach day that samples were tested, and buffers were adjustedaccordingly.

20 μl of the resolution solution was injected, and the HPLC program wasrun. Desamido-hGH appeared as a small peak at a retention time of about0.85 relative to the principal peak. The test was not valid unless theresolution between the peaks corresponding to hGH and desamido-hGH wasat least 1.0 and the symmetry factor of the hGH peak was 0.8 to 1.8. 20μl of the test article was injected, and the HPLC program was run.Samples were run in triplicate. Average retention times were reported.

Modifications to various conditions and/or parameters may be required toanalyze PEGylated hGH and other forms. Modifications to RP-HPLC areknown to those of ordinary skill in the art.

Data Analysis

The average retention time of the test article was compared with thestandard. The average purity of the test article was calculated:Integration area of the main peak/integration areas of all peaks)×100%.Any peak(s) due to the solvent were disregarded.

Results from RP-HPLC for MetY35pAF hGH with the WHO hGH standard areprovided in Table 3 and FIG. 17. Specifications such as the retentiontime difference between the test article and the reference standard andpurity may vary with different molecules or forms of a molecule (i.e.Y35pAF hGH compared to metY35pAF hGH).

TABLE 3 Ret Time % (min) Area Area Symmetry Resolution WHO Main Peak34.4 36576 99.3 0.86 Deamidated WHO Deam. Peak 28.069 4920 11.05 Mainpeak 33.746 38180 85.76 1.54 MetY35pAF Main peak 42.307 31432 96.08 0.69Deamidated MetY35pAF Deamid. peak 34.461 5225 16.67 Main peak 42.03424534 78.28 1.72 MetY35pAF(2) Main peak 41.938 30304 96.19 0.69Deamidated MetY35pAF (2) Deamid. peak 34.209 5039 16.74 Main peak 41.49523716 78.82 1.69

Example 5 Size Exclusion Chromatography

Size-exclusion high performance liquid chromatography (SEC-HPLC) is atechnique using the stationary phase as a porous matrix which ispermeated by mobile phase molecules. Sample molecules small enough toenter the pore structure are retarded, while larger molecules areexcluded and therefore rapidly carried through the column. Thus, sizeexclusion chromatography provides separation of molecules by size andthe chromatographic elution time is characteristic for a particularmolecule.

SEC-HPLC was used to assess recombinant human growth hormone (hGH)potency. SEC-HPLC was a method to determine the percentage of monomer(PEGylated and non-PEGylated) hGH. Dimer and other high molecular weightproteins were observable using this technique. Thus, this techniqueseparates monomer from dimer and other higher molecular weightsubstances in the sample, as well as PEGylated and non-PEGylated forms.References for this technique, include, but are not limited to, EuropeanPharmacopoeia 2002, p. 193; British Pharmacopoeia 2001, p. 1941; and“High-Performance Size-Exclusion Chromatographic Determination of thePotency of Biosynthetic Human Growth Hormone Products” by R. M. Rigginet al. Journal of Chromatography 435 (1988), p. 307-318.

Equipment for this procedure included, the following or equivalentsthereof: a UV/Vis Spectrophotometer (Agilent 8453 or equivalent); 50 ulquartz cuvette; 0.5 mL Vivaspin concentrators (if needed; Vivascience10,000 MWCO, PES, VS0102 or equivalent); HPLC vials and caps (Alltech100 ul screw cap polypropylene vials #12962, TFE liner caps #73048, openhole screw caps #73044, or equivalent); clean 1 and 2 L glass bottles; acolumn such as Tosohaas TSK Super SW3000 18675 and Super SW Guard Column18762, a silica-based size exclusion HPLC column with a dimension of4.6×300 mm, particle size of 4 μm and pore size of 250 Å along with aguard column having a dimension of 4.6×35 mm and 4μ particle size); andHigh-pressure liquid chromatography instrument capable of performinglinear gradients (such as Agilent 1100 HPLC equipped with a vacuumdegasser, quaternary pump, thermostatted autosampler, thermostattedcolumn compartment, diode array detector (DAD), Refractive Indexdetector (RID) and Chemstation chromatography software).

Reagents for this procedure included solid chemicals that wereanalytical grade or better and solvents that were HPLC grade or better,unless otherwise noted. Examples of such chemicals include, MonobasicSodium Phosphate, Spectrum U.S.P. grade S0130, or equivalent; DibasicSodium Phosphate, Spectrum U.S.P. grade S0140, or equivalent;2-propanol, Fisher HPLC grade A451-4, or equivalent. Additionalsolutions include: mobile phase solution (97% of 63 mM sodium phosphatepH 7.0; 3% of 2-propanol) and solution A. To make mobile phase solution,26.8 g of dibasic sodium phosphate was dissolved in 1 L Milli-Q H₂O, and13.79 g monobasic sodium phosphate was dissolved in 1 L Milli-Q H₂O. Thetwo solutions were mixed to give a sodium phosphate buffer of pH 7.0.The 100 mM sodium phosphate, pH 7.0 buffer was diluted to 63 mM withMilli-Q H₂O. 970 mL of 63 mM sodium phosphate pH 7.0 was mixed with 30mL of 2-propanol (or other appropriate volumes to obtain 97% of 63 mMsodium phosphate pH 7.0, 3% of 2-propanol). The resulting buffer wassterile filtered using 0.22 μm PES filters (Corning #431098, orequivalent). Solution A is 25 mM Sodium Phosphate, pH 7.0. 250 mL of 100mM sodium phosphate, pH 7.0 buffer was diluted with 750 mL Milli-Q H₂O.The resulting buffer was sterile filtered using 0.22 μm PES filters(Corning #431098, or equivalent).

Samples for use as standards in SEC-HPLC include World Health,Organization (WHO) hGH (Cat. #98/574) reconstituted with 1.0 ml of waterand diluted to 0.9-1.1 mg/ml. Other hGH reference standards may be usedat 0.9-1.1 mg/ml concentration. For the resolution solution, the hGHstandard was incubated at 50° C. for 12-24 hours, dissolved in solutionA, then diluted to 1 mg/ml with solution A, and stored at −80° C. untiluse. The test material was diluted to 1.0 mg/ml with Solution A. Sampleconcentrations were measured using standard techniques.

Procedure

The instrument was set-up with the following conditions: 1) Column: TSKSuper SW3000 18675 and Guard Column 18762 Auto sampler; 2) Temperature:room temperature; 3) Pump Setup—gradient: isocratic; flow rate: 0.3ml/min; duration: 25 min; Max Pressure: 120 bar; 4) Injectorsetup—Injection: Standard Injection; Injection Volume: 20 μl; DrawSpeed: 100 μl/min; Injection Speed: 100 μl/min; Needle wash: 100 ul H₂O;Stop Time: As pump; 4) DAD Signals—Table 4; Peak Width >0.05 min; Slit:2 nm; Stop Time: As pump;

TABLE 4 Sample Bw Reference Bw Units 214 4 600 100 nm 276 4 600 100 nm220 8 600 100 nm 280 4 600 100 nm 250 8 600 100 nm5) RID Signal—Temperature: 35° C.; Response Time: >0.2 min 4 s,standard; 6) Column Thermostat: Temperature: 23° C.; Store: Temperature.

The column was equilibrated with 10 column volumes (50 ml=166 minutes at0.3 ml/minutes) of the mobile phase, and the RID was purged for 20minutes before injecting the samples. 20 μl of the resolution solutionwas injected. In the chromatogram obtained, 1) the main non-PEGylatedpeak eluted at a retention time of approximately 13-13.5 minutes, 2) thepeaks corresponding to the hGH dimer eluted at a retention time ofapproximately 12.2-12.6 minutes, and 3) the higher molecular weightproteins (non-PEGylated) eluted at relative retention time of 7.4-8.0minutes. The main PEGylated peak eluted at a retention time ofapproximately 8.3-8.8 minutes. 20 μl of the standard was injected, andthe HPLC program was run. 20 μl of the test article was injected, andthe HPLC program was run. Samples were run in triplicate. Averageretention times were recorded.

Modifications to various conditions and/or parameters may be required tofurther characterize PEGylated hGH or other forms. Modifications toSEC-HPLC are known to those of ordinary skill in the art.

Data Analysis

The Retention Time of the hGH test article is compared with thestandard. For purity determinations of non-PEGylated hGH, the integratedmain peak areas of the hGH test article and the standard is compared,and the percentage of monomer in the hGH test article is calculated:(peak area of hGH sample/peak area of standard)×100%. The percentage ofdimer and/or higher aggregates in the hGH test article is alsocalculated. For purity determinations of PEGylated hGH, the integratedmain peak areas of the PEGylated hGH sample and the standard iscompared, and the percentage of PEGylated monomer in PEGylated-hGHsample is calculated by: (peak area of PEGylated hGH sample/peak area ofstandard)×100%. The percentage of PEGylated dimer, higher aggregates,and non-PEGylated monomer in the hGH test article are calculated also.Alternatively, purity is determined for non-PEGylated hGH or PEGylatedhGH by: (Integration area of the main peak of hGH sample/integrationareas of all peaks of hGH sample)×100%. Any peak(s) due to the solventwere disregarded. Purity determinations are also calculated usingabsolute mass or direct peak area rather than by percent of a referencestandard.

Specifications: The retention time difference between the hGH testarticle and the standard is approximately <±30 seconds. For dimer andhigher molecular weight proteins, in the chromatogram obtained with thetest article, the sum of the areas of any peak with a retention timeless than that of the principal peak is not greater than 4.0% or 6.0% ofthe total area of the peaks, respectively. Any peak(s) due to thesolvent are disregarded.

FIG. 18 shows SEC-HPLC data from 30K PEG-metY35pAF and metY35pAF.

Example 6 Cation-Exchange High Performance Liquid Chromatography(cIEX-HPLC)

Cation-exchange high performance liquid chromatography (cIEX-HPLC) is atechnique that relies on charge-charge interactions between a proteinand the charges immobilized on the resin. Cation exchange chromatographytakes advantage of the positively charged ions of a protein that bind tothe negatively charged resin. A common structural modification of hGH isdeamidation of asparagine (Asn) residues, and this cIEX-HPLC methodpermits the separation of deamidated and deamidation intermediates ofPEGylated and non-PEGylated hGH.

The following method was used to assess relative purity and potentialchemical degradation (i.e. deamidation) of recombinant human growthhormone (hGH). This method was used to support identification and purityassessment of PEGylated and non-PEGylated hGH. Some partial degradationproducts of hGH were observable using this technique.

Equipment for this procedure included, the following or equivalentsthereof: UV/Vis Spectrophotometer (Agilent 8453 or equivalent); 50 ulquartz cuvette; 0.5 mL Vivaspin concentrators (if needed; Vivascience10,000 MWCO, PES, VS0102 or equivalent); HPLC vials and caps (Alltech100 ul screw cap polypropylene vials #12962, TFE liner caps #73048, openhole screw caps #73044, or equivalent); clean 1 and 2 L glass bottles; acolumn such as PolyCAT A 4.6×200 mm, 5μ, 1000 Å (204CT0510) and PolyCATA guard column, 4.6×10 mm, 5μ, 1000 Å (JGCCT0510); and High-pressureliquid chromatography instrument capable of performing linear gradients(such as Agilent 1100 HPLC equipped with a vacuum degasser, quaternarypump, thermostatted autosampler, thermostatted column compartment, diodearray detector (DAD), Refractive Index detector (RID) and Chemstationchromatography software).

Reagents for this procedure included solid chemicals that wereanalytical grade or better and solvents that were HPLC grade or better,unless otherwise noted. Examples of such chemicals include, AmmoniumAcetate, Spectrum HPLC grade A2149, or equivalent; Acetonitrile, FisherHPLC grade A998, or equivalent, Ammonium Bicarbonate. Additionalsolutions include: Mobile phase A (40% Acetonitrile, H₂O); Mobile phaseB (40% Acetonitrile, 500 mM Ammonium Acetate, pH 4.5) and Buffer forDeamidation Control (30 mM Ammonium Bicarbonate, pH 9.0). For Mobilephase A buffer, 400 mL HPLC grade Acetonitrile was mixed with 600 mlsterile filtered Milli-Q H₂O, and the resulting mixture was sterilefiltered using 0.22 μm PES filters (Corning #431098), or equivalent).For Mobile phase B buffer, 38.54 g ammonium acetate was dissolved in 970mL Milli-Q H₂O. The solution was brought to pH 4.5 with glacial aceticacid, and then the volume brought up to 1000 mL volume with Milli-Q H₂O.The buffer was sterile filtered using 0.22 μm PES filters (Corning#431098). 100 mL Milli-Q H₂O, 400 mL HPLC grade Acetonitrile, and 500 mL500 mM Ammonium Acetate, pH 4.5 were mixed. For the Buffer forDeamidation Control, 2.37 g of Ammonium bicarbonate was dissolved in0.95 L Milli-Q H₂O, and the pH was brought to 9.0 with NaOH. The volumewas then brought up to 1 L with Milli-Q H₂O. The resulting buffer wassterile filtered using 0.22 μm PES filters (Corning #431098, orequivalent).

Samples for use as standards in cIEX-HPLC included World Health,Organization (WHO) hGH (Cat. #98/574) reconstituted with 1.0 ml of waterand diluted to 0.9-1.1 mg/ml. Other hGH reference standards may be usedat 0.9-1.1 mg/ml concentration. For the resolution solution, hGHstandard(s) were buffer exchanged into 30 mM Ammonium Bicarbonate, pH9.0 buffer using a PD-10, Nap-10, or Nap-5 desalting column (dependingon sample volume). The standard was concentrated using a 0.5 mL Vivaspinconcentrator to 0.9-1.1 mg/ml, incubated at 37° C. for 24 hours, andthen stored at −80° C. until use. The test article was diluted to 1.0mg/ml. A deamidated resolution standard was included. Alternatively, thetest article and standards are diluted over a range of concentrations.Sample concentrations were measured using standard techniques.

Procedure

The instrument was set-up with the following conditions: 1) Column:PolyCAT A 204CT0510 and JGCCT0510; 2) Auto sampler Temperature: 4° C.;3) Pump Setup—gradient: 50 50-250 mM Ammonium Acetate pH 4.5;

TABLE 5 Flow Pressure Time Mobile Phase A Mobile Phase B (ml/min) (bar)0 90 10 0.5 140 14 90 10 0.5 140 79 50 50 0.5 140 19.5 50 50 0.75 140 800 100 1 140 115 0 100 1 140 115.5 90 10 1 140 150 90 10 1 140 150.5 9010 0.5 140 153 90 10 0.5 1404) Injector Setup—Injection: Standard Injection; Injection Volume: 15μl; Draw Speed: 50 μl/min; Injection Speed: 50 μl/min; Needle wash: 15ul H₂O; Stop Time: As pump; 5) DAD Signals—Table 5; Peak Width: >0.1min; Slit: 4 nm; Stop Time: As pump;

TABLE 5 Sample Bw Reference Bw Units 280 4 600 100 nm 276 4 600 100 nm214 8 600 100 nm 220 4 600 100 nm 250 8 600 100 nm

6) Column Thermostat: Temperature: 20° C.; Store: Temperature.

The column was equilibrated with 10 column volumes of 90% mobile phase Aand 10% mobile phase B. 15 μl of the resolution solution was injected.In the chromatogram obtained, the main non-PEGylated peak elutes at aretention time of approximately 64-67 minutes, the non-PEGylateddeamidated peak elutes at a retention time of approximately 62-65minutes, the main PEGylated peak elutes at a retention time ofapproximately 43-46 minutes, and the PEGylated deamidated peak elutes ata retention time of approximately 41-44 minutes. 15 μl of the standardwas injected, and the HPLC program was run. 15 μl of the test articlewas injected, and the HPLC program was run. Samples were run intriplicate. Average retention times were reported.

Modifications to various conditions and/or parameters may be required toanalyze PEGylated hGH and other forms. Modifications to cIEX-HPLC areknown to those of ordinary skill in the art. Additional referencestandards include, but are not limited to, in-process pY35pAF forin-process release of bulk pY35pAF, purified pY35pAF for quantifyingresidual free pY35pAF in PEGylated-pY35pAF, and purified PEG-pY35pAF.

Data Analysis

The retention times of the hGH test article with the standard werecompared and purity determinations of non-PEGylated hGH and PEGylatedhGH calculated. Purity may be determined by calculations such as:(Integration area of the main peak of hGH sample/integration areas ofall peaks of hGH sample)×100%. Any peak(s) due to the solvent weredisregarded.

Table 6 and FIG. 19 show cIEX-HPLC analysis of 30K PEGmY35pAF hGH andmY35pAF hGH.

TABLE 6 Deamid 30K 30K PEG Rel. PEG Rel. Deamid Rel Rel mY35pAF %mY35pAF % mY35pAF % mY35pAF % mY35pAF 66.26 100 Deamid 63.48 18.5 81.4mY35pAF 30K PEG 43.37 5.3 45.07 72.1 mY35pAF Deamid 43.32 21.1 45.4264.9 30K PEG mY35pAF

Example 7

This example describes the synthesis of p-Acetyl-D,L-phenylalanine (pAF)and m-PEG-hydroxylamine derivatives.

The racemic pAF was synthesized using the previously described procedurein Zhang, Z., Smith, B. A. C., Wang, L., Brock, A., Cho, C. & Schultz,P. G., Biochemistry, (2003) 42, 6735-6746.

To synthesize the m-PEG-hydroxylamine derivative, the followingprocedures were completed. To a solution of (N-t-Boc-aminooxy)aceticacid (0.382 g, 2.0 mmol) and 1,3-Diisopropylcarbodiimide (0.16 mL, 1.0mmol) in dichloromethane (DCM, 70 mL), which was stirred at roomtemperature (RT) for 1 hour, methoxy-polyethylene glycol amine(m-PEG-NH₂, 7.5 g, 0.25 mmol, Mt. 30 K, from BioVectra) andDiisopropylethylamine (0.1 mL, 0.5 mmol) were added. The reaction wasstirred at RT for 48 hours, and then was concentrated to about 100 mL.The mixture was added dropwise to cold ether (800 mL). Thet-Boc-protected product precipitated out and was collected by filtering,washed by ether 3×100 mL. It was further purified by re-dissolving inDCM (100 mL) and precipitating in ether (800 mL) twice. The product wasdried in vacuum yielding 7.2 g (96%), confirmed by NMR and Nihydrintest.

The deBoc of the protected product (7.0 g) obtained above was carriedout in 50% TFA/DCM (40 mL) at 0° C. for 1 hour and then at RT for 1.5hour. After removing most of TFA in vacuum, the TFA salt of thehydroxylamine derivative was converted to the HCl salt by adding 4N HClin dioxane (1 mL) to the residue. The precipitate was dissolved in DCM(50 mL) and re-precipitated in ether (800 mL). The final product (6.8 g,97%) was collected by filtering, washed with ether 3×100 mL, dried invacuum, stored under nitrogen. Other PEG (5K, 20K) hydroxylaminederivatives were synthesized using the same procedure.

Example 8

This example describes expression and purification methods used for hGHpolypeptides comprising a non-natural amino acid. Host cells have beentransformed with orthogonal tRNA, orthogonal aminoacyl tRNA synthetase,and hGH constructs.

A small stab from a frozen glycerol stock of the transformed DH10B(fis3)cells were first grown in 2 ml defined medium (glucose minimal mediumsupplemented with leucine, isoleucine, trace metals, and vitamins) with100 μg/ml ampicillin at 37° C. When the OD₆₀₀ reached 2-5, 60 μl wastransferred to 60 ml fresh defined medium with 100 μg/ml ampicillin andagain grown at 37° C. to an OD₆₀₀ of 2-5. 50 ml of the culture wastransferred to 2 liters of defined medium with 100 μg/ml ampicillin in a5 liter fermenter (Sartorius BBI). The fermenter pH was controlled at pH6.9 with potassium carbonate, the temperature at 37° C., the air flowrate at 5 lpm, and foam with the polyalkylene defoamer KFO F119(Lubrizol). Stirrer speeds were automatically adjusted to maintaindissolved oxygen levels ≧30% and pure oxygen was used to supplement theair sparging if stirrer speeds reached their maximum value. After 8hours at 37° C., the culture was fed a 50× concentrate of the definedmedium at an exponentially increasing rate to maintain a specific growthrate of 0.15 hour⁻¹. When the OD₆₀₀ reached approximately 100, a racemicmixture of para-acetyl-phenylalanine was added to a final concentrationof 3.3 mM, and the temperature was lowered to 28° C. After 0.75 hour,isopropyl-b-D-thiogalactopyranoside was added to a final concentrationof 0.25 mM. Cells were grown an additional 8 hour at 28° C., pelleted,and frozen at −80° C. until further processing.

The His-tagged mutant hGH proteins were purified using the ProBondNickel-Chelating Resin (Invitrogen, Carlsbad, Calif.) via the standardHis-tagged protein purification procedures provided by Invitrogen'sinstruction manual, followed by an anion exchange column.

The purified hGH was concentrated to 8 mg/ml and buffer exchanged to thereaction buffer (20 mM sodium acetate, 150 mM NaCl, 1 mM EDTA, pH 4.0).MPEG-Oxyamine powder was added to the hGH solution at a 20:1 molar ratioof PEG:hGH. The reaction was carried out at 28° C. for 2 days withgentle shaking. The PEG-hGH was purified from un-reacted PEG and hGH viaan anion exchange column.

Example 9 Purity Analysis by SDS-PAGE

The following method was used to evaluate the purity of PEG-recombinanthGH conjugates by SDS-PAGE, followed by total protein staining. Anycharged molecule such as a protein will migrate when placed in anelectric field. The velocity of migration of a protein in an electricfield depends on the strength of the electric field, the net electriccharge on the protein, and the frictional resistance. The frictionalresistance is the function of the size and shape of the protein. Whendenatured in the presence of excess SDS, most proteins bind SDS in aconstant weight ratio such that they have essentially identical chargedensities and migrate in polyacrylamide gels according to protein size.Proteins separated by gel electrophoresis can be detected by CoomassieBrilliant Blue staining.

Equipment for this procedure included, the following or equivalentsthereof: XCell Surelock Mini-Cell (Invitrogen), heat block set to+70-80° C., power supply (up to 200V), microcentrifuge (such as BeckmanCoulter Microfuge 18 or 22R), and reciprocal shaker. Reagents includedNuPAGE MOPS SDS Running Buffer (20×, Invitrogen PN NP0001); NuPAGE MESSDS Running Buffer (20×, Invitrogen PN NP0002); NuPAGE LDS Sample Buffer(4×, Invitrogen PN NP0007); NuPAGE Sample Reducing Agent (10×,Invitrogen PN NP0009); 12% Bis-Tris NuPAGE precast gel, 1.0 mm×10-well(Invitrogen PN NP0341BOX); 4-12% Bis-Tris NuPAGE precast gel, 1.0mm×10-well (Invitrogen PN NP0321BOX); Pre-Stained Molecular WeightMarker (SeeBlue Plus2, Invitrogen PN LC5925); MilliQ-quality H₂O orequivalent; SimplyBlue SafeStain (Invitrogen PN LC6065) or equivalent;reference standard (WHO rhGH standard; calibration solutions for rhGH(Y35pAF-pB2/pB3, 2 mg/ml); calibration solutions for the pEG-rhGHconjugate (PEG30-pY35pAF-01, 2 mg/mL). Protein concentrations of thestandards and the test article were measured using standard techniquesknown in the art.

Analysis of PEGylated rhGH Product

10 μg of the reference standard (RS, e.g. calibration solutionPEG30-pY35pAF-01) was prepared under non-reducing and reducingconditions. 10 ug of PEG30-pY35pAF-01 (2 mg/mL) was added to 4×LDS andMilliQ H₂O to obtain a final 28 μl sample in 1×LDS. For reducedconditions, 10 μg of reference standard was added to 4×LDS, 10× ReducingAgent, and MilliQ H₂O to obtain a 28 μl sample in 1×LDS and 1× ReducingAgent. Similarly, 10 μg of PEGylated rhGH test articles were alsoprepared under non-reduced and reduced conditions. The PEG-rhGH testarticles and PEG-rhGH reference standards were not heated, but were snapcentrifuged prior to loading on 4-12% Bis-Tris NuPAGE precast gelsprepared with 1×MES SDS Running Buffer according to manufacturer'sinstructions. The gels were loaded in the order of Pre-Stained MolecularWeight Marker, 10 μg reference standard, blank lane (recommended tominimize potential carryover effects), followed by the test articleswith a maximum setting of 200V for 35 minutes. The gels were incubatedin di-H₂O, stained with shaking using SimplyBlue or an equivalent, anddestained with water.

The electropherogram of the PEG-rhGH test article should conform to theelectropherogram obtained with the PEG-rhGH reference standard. Anybands that do not match the reference standard may be degradationproducts or aggregates. Higher molecular weight bands may representaggregates, and lower molecular weight bands may represent polypeptidethat is no longer conjugated to PEG.

Example 10 Purity and Chemical Degradation Analysis of rhGH byCEX-HPLC/IEX-HPLC

The following method was used to assess relative purity and potentialchemical degradation (i.e. deamidation) of PEGylated recombinant humangrowth hormone (rhGH) by cation-exchange high performance liquidchromatography (CEX-HPLC). CEX-HPLC is a technique that relies oncharge-charge interactions between a protein and the charges immobilizedon the resin. Cation exchange chromatography takes advantage of thepositively charged ions of a protein that bind to the negatively chargedresin. A common structural modification of rhGH deamidation ofasparagine (Asn) residues, and this CEX-HPLC method permits theseparation of deamidated and deamidation intermediates of PEGylated andnonPEGylated rhGH. This method was used to support identification andpurity assessment of PEGylated rhGH. Some partial degradation productsof rhGH are observable using this technique.

Equipment for this procedure included, the following or equivalentsthereof: UV/Vis Spectrophotometer (Agilent 8453 or equivalent); 50 μlquartz cuvette; 0.5 mL Vivaspin concentrators (if needed; Vivascience10,000 MWCO, PES, VS0102 or equivalent); PD-10, NAP-10, or NAP-5 column(GE Healthcare, Cat. #17-0851-01, 17-0853-01, 17-0854-01); HPLC vialsand caps (Alltech 100 μl screw cap polypropylene vials #12962, TFE linercaps #73048, open hole screw caps #73044, or equivalent); clean 1 and 2L glass bottles; column-PolyCAT A 4.6×200 mm, 5μ, 1000 Å (PolyLC,204CT0510) and PolyCAT A guard column, 4.6×10 mm, 5μ, 1000 Å (PolyLC,JGCCT0510); high-pressure liquid chromatography instrument capable ofperforming linear gradients (such as Agilent 1100 HPLC equipped with avacuum degasser, quaternary pump, thermostatted autosampler,thermostatted column compartment, diode array detector (DAD), andChemstation chromatography software).

Reagents for this procedure included water (Milli-Q quality orequivalent) and solid chemicals are analytical grade or better andsolvents are HPLC grade or better, unless otherwise noted. Storage ofreagents and procedural steps occurred at room temperature, unlessotherwise indicated. Examples of such chemicals include AmmoniumAcetate, Spectrum A2149, HPLC grade, or equivalent; Acetonitrile, FisherA998, HPLC grade, or equivalent; Ammonium Bicarbonate, Fluka #09830,Ultra >99.5%, or equivalent; Glacial Acetic Acid, Fisher #64-19-7, HPLCgrade, or equivalent; Sodium Citrate Dihydrate, Spectrum S0165, USPgrade, or equivalent; Glycine, Spectrum AM125 or equivalent; Mannitol,Spectrum MA165, or equivalent; 6N HCl, Mallinckrodt 2662-46, orequivalent.

Mobile phase A buffer was 50 mM Ammonium Acetate, pH 4.25, 40%Acetonitrile (AcCN), and Mobile Phase B buffer was 500 mM AmmoniumAcetate, pH 4.25, 40% AcCN. Additional reagents prepared were 10% aceticacid; Buffer for Deamidation: 30 mM Ammonium Bicarbonate, pH 9.0; andSample Dilution Buffer: 20 mM Sodium Citrate, 20 g/L Glycine, 5 g/LMannitol, pH 6.0, each sterile filtered using 0.22 μm PES filters(Corning #431098, or equivalent).

World Health Organization (WHO) rhGH (Cat. #98/574) was used as anon-PEGylated hGH standard. It was reconstituted in 1.0 ml of water anddiluted to 1.1 mg/ml using dilution buffer. 10% (v/v) of 10% acetic acidwas added to bring the pH between pH 3.8-4.3 with a final concentrationof 1.0 mg/ml (acceptable range 0.9-1.1 mg/ml). Another non-PEGylated hGHstandard, the calibration solution Y35pAF-pB2/pB3, was prepared in asimilar fashion. A PEGylated hGH standard, calibration solutionPEG30-pY35pAF-01, was also prepared in a similar fashion.

For the PEGylated Resolution Solution, the PEG30-pY35pAF-01 calibrationsolution was buffer exchanged into 30 mM Ammonium Bicarbonate, pH 9.0buffer using a PD-10, Nap-10, or Nap-5 desalting column. The standardwas concentrated using a 0.5 mL Vivaspin concentrator to approximately 2mg/ml (acceptable range 1.9-2.1 mg/ml), and the sample was incubated at37° C. for 24 hours. The sample or portion of the sample needed wasdiluted to 1.1 mg/ml using dilution buffer, and 10% (v/v) of 10% aceticacid was added to bring pH between pH 3.8-4.3 with a final concentrationof 1.0 mg/ml (acceptable range 0.9-1.1 mg/ml).

The test article was diluted to 1.1 mg/ml using dilution buffer and 10%(v/v) of 10% acetic acid was added to bring pH between pH 3.8-4.3 with afinal concentration of 1.0 mg/ml (acceptable range 0.9-1.0 mg/ml).Protein concentrations of the standards and the test article weremeasured using standard techniques known in the art.

Procedure

The instrument was set-up with the following conditions: 1) Column:PolyCAT A 204CT0510 and JGCCT0510; 2) Auto sampler Temperature: roomtemperature; 3) Pump Setup: step gradient: 81.5-108.5 mM AmmoniumAcetate pH 4.25 (7-13% B), followed by 108.5-500 mM Ammonium Acetate pH4.25 (13-100% B); 4) Table 7;

TABLE 7 Flow Pressure Time Mobile Phase A Mobile Phase B (ml/min) (bar)0 100 0 1.0 140 10 100 0 1.0 140 11 93 7 1.0 140 91 87 13 1.0 140 102 0100 1.0 140 118 0 100 1.0 140 119 100 0 1.0 140 151 100 10 1 1405) Injector Setup—Injection: Standard Injection; Injection Volume: 25μl; Draw Speed: 50 μl/min; Injection Speed: 50 μl/min; Needle wash: 15μl H₂O; Stop Time: As pump; 6) DAD signals: Table 8;

TABLE 8 Sample Bw Reference Bw Units 280 4 600 100 nm 276 4 600 100 nm214 8 600 100 nm 220 4 600 100 nm 250 8 600 100 nmPeak Width: >0.1 min; Slit: 4 nm; Stop Time: as pump; 7) ColumnThermostat: Temperature: 30° C.; record the temperature.

The column was equilibrated with 10-15 column volumes of 100% mobilephase A. 25-50 μl of the PEGylated calibration solution PEG30-pY35pAF-01was injected. The main PEGylated peak eluted at a retention time of56.97 min (±0.5 min). Next, 25-50 μl of the WHO or calibration solutionY35pAF-pB2/pB3 was injected and the HPLC program was run. The mainnon-PEGylated peak eluted at a retention time of 98.54 min (±0.5 min), arelative retention time of 1.73±0.01 to the main PEGylated peak.

25-50 μl of the PEGylated resolution solution was then injected. In thechromatogram obtained, the main PEGylated peak eluted at a retentiontime of 56.97 min (±0.5 min), and the PEGylated deamidated peak elutedat a retention time of 0.79±0.02 relative to the main peak (45.23±0.3min; (current conditions result in a resolution of 2.3±0.02).

25-50 μl of the PEGylated test article was then injected, and the HPLCprogram was run. The samples were run in triplicate, and averageretention times were noted. Chromatograms were generated with absorbance(280 nm).

Data Analysis

The retention time of the PEGylated rhGH test article was compared withthe calibration solution PEG30-pY35pAF-01. The average purity of thetest article was calculated using: (Integration area of the mainpeak/integration areas of all peaks)×100%. Any peak(s) due to thesolvent were disregarded.

Example 11 Purity Determination of rhGH by SEC-HPLC

This procedure was used to assess the purity of recombinant human growthhormone (rhGH) and PEGylated rhGH by size-exclusion high performanceliquid chromatography (SEC-HPLC). This test separates monomer from dimerand other related substances of higher molecular weight in the sample,as well as PEGylated and nonPEGylated samples. SEC-HPLC is a techniqueusing the stationary phase as a porous matrix which is permeated bymobile phase molecules. Sample molecules small enough to enter the porestructure are retarded, while larger molecules are excluded andtherefore rapidly carried through the column. Thus, size exclusionchromatography means separation of molecules by size and thechromatographic elution time is characteristic for a particularmolecule. This procedure is used to determine the percentage of monomer(PEGylated and unPEGylated) rhGH. Dimer and other high molecular weightproteins are observable using this technique. An example of SEC-HPLCintegration is shown as FIG. 20 with the y-axis as absorbance (214 nm)and the x-axis as time (minutes).

References for this technique include European Pharmacopoeia 2002, p.193; British Pharmacopoeia 2001, p. 1941; “High-PerformanceSize-Exclusion Chromatographic Determination of the Potency ofBiosynthetic Human Growth Hormone Products” by R. M. Riggin et al.Journal of Chromatography 435 (1988), p. 307-318.

Equipment for this procedure included, the following or equivalentsthereof: UV/Vis Spectrophotometer (Agilent 8453 or equivalent); 50 ulquartz cuvette; 0.5 mL Vivaspin concentrators (if needed; Vivascience10,000 MWCO, PES, VS0102 or equivalent); HPLC vials and caps (Alltech100 ul screw cap polypropylene vials #12962, TFE liner caps #73048, openhole screw caps #73044, or equivalent); clean 1 and 2 L glass bottles;Column—Tosohaas TSK Super SW3000 18675 and Super SW Guard Column 18762,a silica-based size exclusion HPLC column with a dimension of 4.6×300mm, particle size of 4 μm and pore size of 250 Å along with a guardcolumn having a dimension of 4.6×35 mm and 4μ particle size;High-pressure liquid chromatography instrument capable of performinglinear gradients (such as Agilent 1100 HPLC equipped with a vacuumdegasser, quaternary pump, thermostatted autosampler, thermostattedcolumn compartment, diode array detector (DAD), Refractive Indexdetector (RID) and Chemstation chromatography software).

Reagents for this procedure included water (Milli-Q quality orequivalent) and solid chemicals are analytical grade or better andsolvents are HPLC grade or better, unless otherwise noted. The storageof reagents and procedural steps occurred at room temperature, unlessotherwise indicated. Examples of such chemicals included MonobasicMonohydrate Sodium Phosphate, Spectrum U.S.P. grade S0130, orequivalent; Dibasic Heptahydrate Sodium Phosphate, Spectrum U.S.P. gradeS0140, or equivalent; 2-propanol, Fisher HPLC grade A451-4, orequivalent.

Mobile phase buffer was 97% of 63 mM sodium phosphate pH 7.0, 3% of2-propanol. Solution A was 25 mM Sodium Phosphate, pH 7.0. Both weresterile filtered using 0.22 μm PES filters (Corning #431098, orequivalent).

World Health Organization (WHO) rhGH (Cat. #98/574) was used as anon-PEGylated hGH standard. It was reconstituted with 1.0 ml of waterand diluted to 1 mg/ml concentration (acceptable range 0.9-1.1 mg/ml) inWHO buffer. Another non-PEGylated hGH standard, calibration solutionY35pAF-pB2/pB3, was prepared in a similar fashion and diluted with 20 mMsodium citrate, 2% glycine, 0.5% mannitol, pH 6. A PEGylated hGHstandard, calibration solution PEG30-pY35pAF-01, was also prepared in asimilar fashion and diluted with 20 mM sodium citrate, 2% glycine, 0.5%mannitol, pH 6. For the Resolution Solution, the PEG30-pY35pAF-02 highermolecular weight standard was brought to 1 mg/ml concentration(acceptable range 0.9-1.1 mg/ml). This solution contains approximately33% PEG-PEG-GH, 66.5% PEG-GH): Test material was diluted toapproximately 1.0 mg/ml with Solution A (acceptable range 0.9-1.1mg/ml). All sample concentrations were measured using standardtechniques known in the art. The dilution of samples may be performedwith any suitable buffer.

Procedure

The instrument was set-up with the following conditions: 1) Column: TSKSuper SW3000 18675 and Guard Column 18762; 2) Pump Setup—gradient:isocratic; flow rate: 0.3 ml/min; duration: 25 min; Max Pressure: 120bar; 3) Injector Setup—Injection: Standard Injection; Injection Volume:10 μl; Draw Speed: 100 μl/min; Injection Speed: 100 μl/min; Needle wash:100 ul H₂O; Stop Time: As pump; 4) DAD Signals: Table 9;

TABLE 9 Sample Bw Reference Bw Units 214 4 600 100 nm 276 4 600 100 nm220 8 600 100 nm 280 4 600 100 nm 250 8 600 100 nmPeak Width: >0.05 min; Slit: 2 nm; Stop Time: as pump; 5) RIDSignal—Temperature: 35° C.; Response Time: >0.2 min 4 s, standard; 6)Column Thermostat: Temperature: 23° C.; record the temperature.

The column was equilibrated with 10 column volumes (50 ml=166 min at 0.3ml/min) of the mobile phase, and the RID was purged for at least 20minutes before injecting samples. DAD and RI detectors were autobalancedbefore sample runs.

20 μl of the calibration solution Y35pAF-pB2/pB3 (or WHO standard) wasinjected, and the HPLC program was run. In the chromatogram obtained,the main unPEGylated peak eluted at a retention time of approximately12.96 (±0.05) min. The higher molecular weight unPEGylated rhGH dimereluted at a retention time of 0.94±0.02 relative to the main peak.Higher molecular weight aggregates eluted at retention times of 7.3-8.0min.

20 μl of the calibration solution PEG30-pY35pAF-01 was injected. Themain pegylated peak eluted at a retention time of approximately 8.33(±0.08) min (relative retention time of 0.64 to the unPEGylated rhGH).Higher molecular weight PEGylated rhGH aggregates eluted at timesgreater than 8.0 min.

20 μl of the resolution solution was injected, and the HPLC program wasrun. The main PEGylated peak elutes at a retention time of 8.28 min, andthe higher molecular weight species eluted at 7.54 min, a relativeretention time of 0.9 (±0.05) relative to the main PEGylated peak.

20 μl of the test article was injected, and the HPLC program was run.Samples were run in triplicate and average retention times were noted.The retention time of the rhGH test article was compared with the rhGHstandard(s).

The SEC-HPLC data from the test article was compared to data obtainedfrom the reference standards. To determine the purity of PEGylated rhGH,the integrated main peak areas of the PEGylated rhGH sample was comparedwith the total peak area, and the percentage of PEGylated monomer inPEG-rhGH sample was calculated by: (main peak area of PEG-rhGHsample/total peak area)×100%. The percentage of PEGylated dimer, higheraggregates, and nonPEGylated monomer were calculated in the PEGylatedhGH test article. Any peak(s) due to the solvent were disregarded. Peakseluting in the chromatogram prior to the main PEGylated hGH peakrepresent higher molecular weight species. Such higher molecular weightspecies may include but are not limited to dimers (such as PEG-PEG-hGHand other possible dimers) or soluble aggregates. Peaks eluting afterthe main PEGylated hGH peak represent lower molecular weight species.Such lower molecular weight species may include but are not limited tonon-PEGylated monomer and clipped forms of PEGylated hGH.

Example 12 Purity and Chemical Degradation Analysis of PEG-hGH byRP-HPLC

The following method was used to assess relative purity and potentialchemical degradation (i.e. oxidation) of PEGylated recombinant humangrowth hormone (PEG-hGH) by reverse phase high performance liquidchromatography (RP-HPLC). RP-HPLC is a technique that separatesmolecules on the basis of relative hydrophobicities. Samples are passedover a stationary phase of silica covalently bonded to hydrocarbonchains. The molecules of interest are retarded by the stationary phaseand eluted with an isocratic solvent. The chromatographic elution timeis characteristic for a particular molecule. This method separatesPEG-hGH from nonPEGylated hGH, as well as isoforms based on subtledifferences in hydrophobicity and retention behavior associated withstructural modifications such as oxidation. This method was used tosupport identification and purity assessment of PEG-hGH. Some partialdegradation products of rhGH are observable using this technique. Anexample of RP-HPLC integration is shown as FIG. 21 with the y-axis asabsorbance (214 nm) and the x-axis as time (minutes).

Equipment for this procedure included, the following or equivalentsthereof: UV/Vis Spectrophotometer (Agilent 8453 or equivalent); 50 μlquartz cuvette; 0.5 mL Vivaspin concentrators (if needed; Vivascience10,000 MWCO, PES, VS0102 or equivalent); HPLC vials and caps (Alltech100 μl screw cap polypropylene vials #12962, TFE liner caps #73048, openhole screw caps #73044, or equivalent); clean 1 and 2 L glass bottles;Column—J. T. Baker Wide Pore Butyl, 250×4.6 mm (cat#7116-00);High-pressure liquid chromatography instrument capable of performinglinear gradients (such as Agilent 1100 HPLC equipped with a vacuumdegasser, quaternary pump, thermostatted autosampler, thermostattedcolumn compartment, diode array detector (DAD), and Chemstationchromatography software).

Reagents for this procedure included water (Milli-Q quality orequivalent) and solid chemicals are analytical grade or better andsolvents are HPLC grade or better, unless otherwise noted. Storage ofreagents and procedural steps occurred at room temperature, unlessotherwise indicated. Examples of such chemicals include, Acetonitrile,Fisher A998, HPLC grade, or equivalent; Trifluoroacetic Acid, HalocarbonUN2699, Biograde, or equivalent; Sodium Phosphate, Spectrum MA1654, USPgrade, or equivalent; Glycine, Spectrum AM125 or equivalent; Mannitol,Spectrum MA165, or equivalent; Trehalose, Fluka 90208, or equivalent;10N Sodium Hydroxide, or equivalent. Mobile Phase A was 0.1% TFA in H₂O,and Mobile phase B was 0.1% TFA in Acetonitrile.

One of the nonPEGylated standards was World Health Organization (WHO)rhGH (Cat. #98/574). It was reconstituted with 1.0 ml of water anddiluted to 1.0 mg/ml (acceptable range 0.9-1.1 mg/ml) with WHO buffer.Another nonPEGylated standard, calibration solution Y35pAF-pB2/pB3, wasprepared in a similar fashion and diluted with 20 mM sodium citrate, 2%glycine, 0.5% mannitol, pH 6. Calibration solution PEG30-pY35pAF-01, aPEGylated standard, was also prepared in a similar fashion and dilutedwith 20 mM sodium citrate, 2% glycine, 0.5% mannitol, pH 6. For thePEGylated Resolution Solution, PEG30-pY35pAF-01 was incubated with 0.1%H₂O₂ for 24 hours at 4° C. to oxidize PEG30-pY35pAF-01. To obtainbetween 5% and 20% oxidized PEG30-pY35pAF-01, this incubation time withH₂O₂ may vary between 4-24 hours. Catalase (20 mg/ml stock) can be addedto stop the oxidation reaction if needed.

The test article was diluted to 1.0 mg/ml using dilution buffer(acceptable range 0.9-1.0 mg/ml). The sample dilution buffer may be anysuitable buffer. Standards and test article protein concentrations weremeasured using standard techniques known in the art.

Procedure

The instrument was set-up with the following conditions: 1) Column: J.T. Baker Wide Pore Butyl, 250×4.6 mm (cat#7116-00); 2) Autosampler—Temperature: 4° C.; 3) Pump Setup: Table 10;

TABLE 10 Time Flow (min) (mL/min) % B 0.0 1.0 31.0 17.5 1.0 64.5 17.751.0 100 18.75 1.0 100 19.0 1.0 31.0 25.0 1.0 31.04) Injector Setup—Injection: Standard Injection; Injection Volume: 10μl; Draw Speed: 100 μl/min; Injection Speed: 100 μl/min; Needle wash: 20μl H₂O; Stop Time: As pump; 5) DAD Signals: Table 11;

TABLE 11 Sample Bw Reference Bw Units 214 4 600 100 nm 276 4 600 100 nm280 16 600 100 nm 220 4 600 100 nm 250 8 600 100 nmPeak Width: >0.1 min; Slit: 4 nm; Stop Time: as pump; 6) ColumnThermostat: Temperature: RT; record the temperature.

New columns were flushed with 10 CV of distilled HPLC-grade water toremove the shipping solvent. The column was cleaned with 10-15 columnvolumes (CV) of 0.1% TFA in 60% isopropanol in MillQ H₂O, and 10-15 CVH₂O. This can be followed by 5 CV of 50:50 DMSO:H₂O if necessary;otherwise the column was equilibrated with mobile phase A.

The column was equilibrated with 10-15 column volumes of 100% mobilephase A. 10 μl of the PEGylated calibration solution PEG30-pY35pAF-01was injected. The main pegylated peak eluted at a retention time of17.05 min (±0.5 min).

10 μl of the WHO or calibration solution Y35pAF-pB2/pB3 was injected,and the HPLC program run. The main nonPEGylated peak elutes at aretention time of 19.04 min (±0.5 min), a relative retention time of1.1±0.05 to the main PEGylated peak.

10 μl of the PEGylated resolution solution was injected. In thechromatogram obtained, the main PEGylated peak eluted at a retentiontime of 17.12 min (±0.5 min), and the PEGylated oxidized peak eluted ata retention time of 0.93±0.02 relative to the main peak (15.94±0.3 minand a resolution of 2.97±0.05).

10 μl of the PEGylated test article was injected, and the HPLC programwas run. Samples were run in triplicate and average retention timesnoted.

The retention time (using A₂₁₄) of the PEGylated rhGH test article wascompared with the calibration solution PEG30-pY35pAF-01. The averagepurity of the test article (using A₂₁₄) was calculated using:(Integration area of the main peak/integration areas of all peaks)×100%.Any peak(s) due to the solvent were disregarded.

Example 13

A summary of the methods used in formulations development is shown asTable 12. Long-term storage, basic, or acidic conditions can result inthe following product-related impurities: Deamidated hGH-Asn149, Asn152;Oxidized hGH-Met14, Met125; Isomerized hGH-Asp130; DehydratedhGH-Asp130; desPhe/des-Phe-Pro—Phe1, Pro2 (non-enzymatic cleavage);Dimer (covalent and non-covalent species); Clipped hGH (cleavage betweenThr142 and Tyr143). Additional methods such as moisture analysis, pH andbioassays including, but not limited to, proliferation assays are usedto evaluate formulations for PEGylated hGH comprising a non-naturallyencoded amino acid.

TABLE 12 desPhe/ Higher Current Structure/ desPhe- MW DePEGylatedMethods Stability Deamidation Oxidation Clipped Isomerization Prospecies ahGH SDS PAGE X X X GEL IEF GEL X X X SEC-HPLC X X RP-HPLC X XCEX-HPLC X X X (X) X Analytical X Ultra- centrifugation for acceleratedstudy Dynamic Light X Scattering for accelerated study FTIR XSDS-PAGE, SEC-HPLC, and cIEX-HPLC show key degradation products.cIEX-HPLC may show cyclic imide, iso-asp (Isomerized hGH-Asp130), anddeamidation intermediates.

Example 14 Lyophilized Formulation Study

Purified PEGylated hGH samples comprising a non-naturally encoded aminoacid in 20 mM sodium citrate, 2% glycine, 0.5% mannitol, pH 6 weredialyzed into a buffer from the formulations matrix (Table 13) prior tostability analysis. A reaction between the non-naturally encoded aminoacid (para-acetylphenylalanine) substituted for a tyrosine at position35 of hGH and mPEG-oxyamine formed an oxime bond between hGH and PEG.The samples were then lyophilized and stored at 4, 25, and 40° C. Afterstorage of the lyophilized samples for various lengths of time (0, 1week, 2 weeks, 4 weeks, 6 weeks, 2 months, and 3 months), the sampleswere reconstituted in water to 2 mg/ml and characteristics such asmoisture content (Karl Fisher), pH and concentration were observed postreconstitution using standard techniques known to one of ordinary skillin the art. The methods described in Examples 9-13 were used to analyzethe PEGylated hGH for degradation products. Sodium phosphate and sodiumsuccinate were included in the formulations matrix. Additional analysesinclude time points at 4 months and 6 months.

TABLE 13 FORMULATIONS MATRIX Formulation Bulking ID Buffer pH AgentStabilizer Antioxidant Surfactant P6MT 10 mM 6 4% Mannitol 2% Trehalosenone 0.01% PS20 Phosphate S4MT 10 mM Succinate 4 4% Mannitol 2%Trehalose none 0.01% PS20 S5MT 10 mM Succinate 5 4% Mannitol 2%Trehalose none 0.01% PS20 S5GT 10 mM Succinate 5 2.5% Glycine 2%Trehalose none 0.01% PS20 H6MT 10 mM Histidine 6 4% Mannitol 2%Trehalose none 0.01% PS20 P6GT 10 mM 6 2.5% Glycine 2% Trehalose none0.01% PS20 Phosphate P6MA 10 mM 6 4% Mannitol 4.4% Arginine none 0.01%PS20 Phosphate P6MS 10 mM 6 4% Mannitol 2% Sucrose none 0.01% PS20Phosphate P6MTMet 10 mM 6 4% Mannitol 2% Trehalose 1 mM 0.01% PS20Phosphate Methionine P7MT 10 mM 7 4% Mannitol 2% Trehalose none 0.01%PS20 Phosphate P7GT 10 mM 7 2.5% Glycine 2% Trehalose none 0.01% PS20Phosphate P6MGT 10 mM 6 2% Mannitol, 2% Trehalose none 0.01% PS20Phosphate 1.25% Glycine P6MGT-P 10 mM 6 2% Mannitol, 2% Trehalose nonePhosphate 1.25% Glycine P6MT-P 10 mM 6 4% Mannitol 2% Trehalose nonePhosphate P6GT-P 10 mM 6 2.5% Glycine 2% Trehalose none Phosphate

Results

Moisture Content, pH, and concentration results are shown in Tables14-32.

TABLE 14 MOISTURE, CONCENTRATION, pH POST RECONSTITUTION; T = 0Formulation Conc. % ID (mg/mL) pH Moisture P6MT 2.1 6.20 0.849 S4MT 2.04.04 0.587 S5MT 2.0 5.02 0.662 S5GT 2.1 5.10 0.495 H6MT 2.1 5.93 0.790P6GT 2.2 6.13 0.442 P6MA 2.1 6.43 1.558 P6MS 2.2 6.11 0.454 P6MTMet 2.16.15 0.713 P7MT 1.9 6.97 0.828 P7GT 2.1 6.94 0.928 P6MGT 2.0 6.14 1.038P6MGT-P 2.0 6.15 4.328 P6MT-P 2.0 6.05 1.522 P6GT-P 2.1 6.10 0.884

TABLE 15 MOISTURE CONTENT Formulation % Moisture % Moisture ID (t = 0)(2 months) P6MT 0.849 0.796 H6MT 0.790 0.714 P6GT 0.442 1.035 P6MS 0.4540.301 P6MTMet 0.713 0.693 P7MT 0.828 0.687 P7GT 0.928 0.670 P6MGT 1.0381.447 P6MGT-P 4.328 1.552 P6MT-P 1.522 1.495 P6GT-P 0.884 0.751

TABLE 16 CONCENTRATION, pH POST RECONSTITUTION; T = 1 week; 4° C.Formulation Conc. ID (mg/mL) pH P6MT 1.7 6.03 S4MT 1.7 3.97 S5MT 1.74.94 S5GT 1.8 5.03 H6MT 1.9 5.88 P6GT 2.0 5.95 P6MA 1.8 6.33 P6MS 1.86.09 P6MTMet 1.8 5.95 P7MT 1.6 6.93 P7GT 1.8 6.89 P6MGT 1.7 6.07 P6MGT-P1.7 5.97 P6MT-P 1.9 5.93 P6GT-P 1.9 5.94

TABLE 17 CONCENTRATION, pH POST RECONSTITUTION; T = 2 week; 4° C.Formulation Conc. ID (mg/mL) pH P6MT 1.7 6.05 S4MT 1.7 4.09 S5MT 1.65.04 S5GT 1.8 5.10 H6MT 1.8 5.98 P6GT 1.9 6.05 P6MA 1.7 6.44 P6MS 1.86.17 P6MTMet 1.8 6.04 P7MT 1.6 6.95 P7GT 1.8 6.91 P6MGT 1.7 6.08 P6MGT-P1.8 6.03 P6MT-P 1.8 6.10 P6GT-P 1.7 6.10

TABLE 18 CONCENTRATION, pH POST RECONSTITUTION; T = 4 week; 4° C.Formulation Conc. ID (mg/mL) pH P6MT 1.6 6.18 S4MT 1.6 3.98 S5MT 1.64.98 S5GT 1.6 5.11 H6MT 1.7 5.98 P6GT 1.8 6.08 P6MA 1.8 6.46 P6MS 1.76.21 P6MTMet 1.8 6.18 P7MT 1.6 7.04 P7GT 1.7 6.96 P6MGT 1.5 6.12 P6MGT-P1.5 6.13 P6MT-P 1.6 6.13 P6GT-P 1.6 6.11

TABLE 19 CONCENTRATION, pH POST RECONSTITUTION; T = 6 week; 4° C.Formulation Conc. ID (mg/mL) pH P6MT 1.7 6.21 S5MT 1.7 5.10 S5GT 1.75.13 H6MT 1.7 6.05 P6GT 1.8 6.12 P6MS 1.7 6.16 P6MTMet 1.8 6.16 P7MT 1.57.01 P7GT 1.8 6.94 P6MGT 1.5 6.18 P6MGT-P 1.6 6.17 P6MT-P 1.7 6.14P6GT-P 1.6 6.18

TABLE 20 CONCENTRATION, pH POST RECONSTITUTION; T = 2 month; 4° C.Formulation Conc. ID (mg/mL) pH P6MT 1.8 6.15 H6MT 1.7 5.99 P6GT 2.06.15 P6MS 1.8 6.15 P6MTMet 1.8 6.15 P7MT 1.5 7.03 P7GT 1.7 6.89 P6MGT1.7 6.09 P6MGT-P 1.6 6.05 P6MT-P 1.7 6.08 P6GT-P 1.7 6.07

TABLE 21 CONCENTRATION, pH POST RECONSTITUTION; T = 3 month; 4° C.Formulation Conc. ID (mg/mL) pH P6MT 1.7 6.16 H6MT 1.9 6.03 P6GT 2.26.14 P6MS 1.9 5.90 P6MTMet 1.9 6.10 P7MT 1.5 7.00 P7GT 1.9 6.93 P6MGT1.7 6.16 P6MGT-P 1.7 6.17 P6MT-P 1.8 6.11 P6GT-P 1.7 6.11

TABLE 22 CONCENTRATION, pH POST RECONSTITUTION; T = 1 week; 25° C.Formulation Conc. ID (mg/mL) pH P6MT 1.7 6.18 S4MT 1.7 4.03 S5MT 1.75.00 S5GT 1.7 5.08 H6MT 1.8 5.90 P6GT 1.9 6.03 P6MA 1.8 6.42 P6MS 1.76.15 P6MTMet 1.7 6.06 P7MT 1.6 6.96 P7GT 1.7 6.89 P6MGT 1.6 6.08 P6MGT-P1.6 6.01 P6MT-P 1.6 6.07 P6GT-P 1.7 6.05

TABLE 23 CONCENTRATION, pH POST RECONSTITUTION; T = 2 week; 25° C.Formulation Conc. ID (mg/mL) pH P6MT 1.7 6.14 S4MT 1.6 4.07 S5MT 1.65.04 S5GT 1.7 5.11 H6MT 1.8 5.96 P6GT 2.0 6.06 P6MA 1.9 6.43 P6MS 1.96.18 P6MTMet 1.9 6.04 P7MT 1.6 6.94 P7GT 1.8 6.91 P6MGT 1.6 6.06 P6MGT-P1.7 6.07 P6MT-P 1.7 6.10 P6GT-P 1.7 6.08

TABLE 24 CONCENTRATION, pH POST RECONSTITUTION; T = 4 week; 25° C.Formulation Conc. ID (mg/mL) pH P6MT 1.6 6.20 S4MT 1.5 4.02 S5MT 1.75.08 S5GT 1.7 5.16 H6MT 1.7 6.02 P6GT 1.7 6.16 P6MA 1.7 6.43 P6MS 1.66.20 P6MTMet 1.8 6.21 P7MT 1.5 7.03 P7GT 1.6 6.98 P6MGT 1.4 6.18 P6MGT-P1.6 6.17 P6MT-P 1.7 6.17 P6GT-P 1.6 6.16

TABLE 25 CONCENTRATION, pH POST RECONSTITUTION; T = 6 week; 25° C.Formulation Conc. ID (mg/mL) pH P6MT 1.7 6.21 S5MT 1.6 5.08 S5GT 1.85.15 H6MT 1.8 6.05 P6GT 1.8 6.14 P6MS 1.8 6.24 P6MTMet 1.8 6.18 P7MT 1.67.00 P7GT 1.7 6.93 P6MGT 1.7 6.13 P6MGT-P 1.7 6.15 P6MT-P 1.7 6.23P6GT-P 1.7 6.18

TABLE 26 CONCENTRATION, pH POST RECONSTITUTION; T = 2 month; 25° C.Formulation Conc. ID (mg/mL) pH P6MT 1.7 6.14 H6MT 1.8 5.94 P6GT 1.96.10 P6MS 1.8 6.15 P6MTMet 1.8 6.07 P7MT 1.5 6.92 P7GT 1.8 6.88 P6MGT1.5 6.09 P6MGT-P 1.7 6.13 P6MT-P 1.7 6.12 P6GT-P 1.7 6.08

TABLE 27 CONCENTRATION, pH POST RECONSTITUTION; T = 3 month; 25° C.Formulation Conc. ID (mg/mL) pH P6MT 1.8 6.17 H6MT 2.1 5.94 P6GT 2.06.12 P6MS 1.9 6.15 P6MTMet 1.9 6.15 P7MT 1.7 6.98 P7GT 1.8 6.92 P6MGT1.7 6.15 P6MGT-P 1.7 6.14 P6MT-P 1.7 6.10 P6GT-P 1.8 6.14

TABLE 28 CONCENTRATION, pH POST RECONSTITUTION; T = 1 week; 40° C.Formulation Conc. ID (mg/mL) pH P6MT 1.8 6.18 S4MT 1.7 4.01 S5MT 1.85.02 S5GT 1.9 5.07 H6MT 1.8 5.92 P6GT 2.0 6.09 P6MA 1.8 6.39 P6MS 1.96.16 P6MTMet 1.7 6.11 P7MT 1.6 6.98 P7GT 1.6 6.93 P6MGT 1.8 6.11 P6MGT-P1.7 6.09 P6MT-P 1.6 6.12 P6GT-P 1.7 6.07

TABLE 29 CONCENTRATION, pH POST RECONSTITUTION; T = 2 week; 40° C.Formulation Conc. ID (mg/mL) pH P6MT 1.6 6.15 S4MT 1.7 4.02 S5MT 1.75.00 S5GT 1.8 5.11 H6MT 1.8 5.93 P6GT 1.9 6.15 P6MA 1.9 6.41 P6MS 1.96.07 P6MTMet 1.8 6.11 P7MT 1.6 6.95 P7GT 1.9 6.90 P6MGT 1.7 6.17 P6MGT-P1.8 6.15 P6MT-P 1.8 6.15 P6GT-P 1.8 6.15

TABLE 30 CONCENTRATION, pH POST RECONSTITUTION; T = 4 week; 40° C.Formulation Conc. ID (mg/mL) pH P6MT 1.6 6.25 S4MT 1.5 3.97 S5MT 1.65.06 S5GT 1.6 5.11 H6MT 1.6 6.01 P6GT 1.7 6.25 P6MA 1.7 6.42 P6MS 1.76.28 P6MTMet 1.6 6.23 P7MT 1.5 7.01 P7GT 1.6 6.98 P6MGT 1.5 6.25 P6MGT-P1.5 6.20 P6MT-P 1.5 6.24 P6GT-P 1.6 6.22

TABLE 31 CONCENTRATION, pH POST RECONSTITUTION; T = 6 week; 40° C.Formulation Conc. ID (mg/mL) pH P6MT 1.7 6.24 S5MT 1.6 5.11 S5GT 1.75.17 H6MT 1.8 6.00 P6GT 1.9 6.17 P6MS 1.8 6.24 P6MTMet 2.5 6.15 P7MT 1.56.98 P7GT 1.8 6.94 P6MGT 1.5 6.28 P6MGT-P 1.7 6.23 P6MT-P 1.7 6.19P6GT-P 1.7 6.22

TABLE 32 CONCENTRATION, pH POST RECONSTITUTION; T = 2 month; 40° C.Formulation Conc. ID (mg/mL) pH P6MT 1.7 6.19 H6MT 1.9 5.99 P6GT 1.96.21 P6MS 1.8 6.19 P6MTMet 1.7 6.18 P7MT 1.5 6.97 P7GT 1.7 6.90 P6MGT1.6 6.15 P6MGT-P 1.6 6.22 P6MT-P 1.7 6.20 P6GT-P 1.7 6.18

SDS gels for this study are shown as FIGS. 22-58. Faint 21.5 kDa bandswere visible in lanes 3 and 4 of FIG. 22.

SEC-HPLC: FIG. 20 is an example SEC-HPLC integration. Tables 33-50 showthe results from this study. The clip peak eluted between the mainPEGylated hGH peak and the Un-PEG peak.

TABLE 33 SEC-HPLC (SUMMARY OF PEAK AREAS); T = 0 Formulation % Pre- %Main Total ID peak Peak Area P6MT 1.38 98.62 4.70E+04 S4MT 2.13 97.874.20E+04 S5MT 1.63 98.37 4.41E+04 S5GT 1.67 98.33 4.58E+04 H6MT 1.6598.35 4.51E+04 P6GT 1.81 98.19 4.66E+04 P6MA 2.09 97.91 4.64E+04 P6MS1.91 98.09 4.54E+04 P6MTMet 1.52 98.48 4.57E+04 P7MT 1.45 98.55 4.14E+04P7GT 1.39 98.61 4.60E+04 P6MGT 1.57 98.43 4.55E+04 P6MGT-P 1.59 98.414.57E+04 P6MT-P 1.88 98.12 4.44E+04 P6GT-P 1.69 98.31 4.52E+04

TABLE 34 SEC-HPLC (SUMMARY OF PEAK AREAS); T = 1 week, 4° C. Formulation% Pre- % Main % Un- Total ID peak Peak PEG Area P6MT 1.81 98.08 0.114.42E+04 S4MT 2.72 96.50 0.78 4.26E+04 S5MT 2.49 97.20 0.32 4.20E+04S5GT 2.17 97.55 0.27 4.58E+04 H6MT 2.23 97.64 0.13 4.71E+04 P6GT 2.5297.48 n/a 4.99E+04 P6MA 2.39 97.44 0.17 4.67E+04 P6MS 2.11 97.71 0.184.83E+04 P6MTMet 2.00 97.83 0.17 4.68E+04 P7MT 1.91 97.90 0.19 4.29E+04P7GT 1.71 98.12 0.17 4.75E+04 P6MGT 1.64 98.15 0.21 4.30E+04 P6MGT-P2.24 97.62 0.14 4.51E+04 P6MT-P 2.30 97.58 0.12 4.54E+04 P6GT-P 2.0497.77 0.18 4.47E+04

TABLE 35 SEC-HPLC (SUMMARY OF PEAK AREAS); T = 2 week, 4° C. Formulation% Pre- % Main % Un- Total ID peak Peak PEG Area P6MT 1.61 98.29 0.104.69E+04 S4MT 2.42 97.16 0.42 4.30E+04 S5MT 1.66 98.26 0.07 4.34E+04S5GT 2.05 97.88 0.08 4.67E+04 H6MT 2.22 97.69 0.09 4.72E+04 P6GT 1.5898.36 0.06 5.11E+04 P6MA 1.52 98.38 0.10 4.95E+04 P6MS 1.47 98.49 0.045.02E+04 P6MTMet 1.61 98.34 0.04 4.98E+04 P7MT 1.53 98.41 0.06 4.46E+04P7GT 1.54 98.41 0.06 4.86E+04 P6MGT 1.48 98.44 0.08 4.59E+04 P6MGT-P1.68 98.23 0.09 4.70E+04 P6MT-P 1.73 98.17 0.10 4.62E+04 P6GT-P 1.5898.39 0.04 4.68E+04

TABLE 36 SEC-HPLC (SUMMARY OF PEAK AREAS); T = 4 week, 4° C. Formulation% Pre- % Pre- % Main % Un- Total ID peak 1 peak 2 Peak PEG Area P6MT1.13 1.30 97.44 0.14 4.54E+04 S4MT 1.18 2.31 95.81 0.69 4.09E+04 S5MT1.62 1.43 96.70 0.25 4.33E+04 S5GT 1.26 1.64 96.81 0.29 4.38E+04 H6MT1.37 1.26 97.24 0.12 4.77E+04 P6GT 1.49 1.60 96.80 0.11 4.76E+04 P6MA1.08 1.44 97.31 0.17 4.89E+04 P6MS 1.56 1.35 96.96 0.13 4.72E+04 P6MTMet1.11 1.29 97.44 0.17 4.88E+04 P7MT 1.07 1.30 97.46 0.17 4.32E+04 P7GT0.83 1.46 97.48 0.23 4.68E+04 P6MGT 1.27 1.32 97.20 0.21 4.09E+04P6MGT-P 1.69 1.24 96.88 0.19 4.38E+04 P6MT-P 1.80 1.38 96.66 0.154.59E+04 P6GT-P 1.90 1.61 96.33 0.16 4.33E+04

TABLE 37 SEC-HPLC (SUMMARY OF PEAK AREAS); T = 6 week, 4° C. Formulation% Pre- % Pre- % Main % Un- Total ID peak 1 peak 2 Peak PEG Area P6MT1.28 1.42 97.20 0.10 4.42E+04 S5MT 2.26 1.57 95.98 0.19 4.28E+04 S5GT1.25 1.81 96.74 0.19 4.44E+04 H6MT 1.23 1.28 97.38 0.10 4.53E+04 P6GT1.94 1.82 96.12 0.12 4.32E+04 P6MS 1.26 1.39 97.25 0.09 4.74E+04 P6MTMet1.19 1.23 97.52 0.06 4.78E+04 P7MT 0.98 1.38 97.55 0.09 4.17E+04 P7GT0.83 1.56 97.46 0.15 4.96E+04 P6MGT 0.97 1.31 97.60 0.12 4.30E+04P6MGT-P 1.37 1.25 97.28 0.11 4.39E+04 P6MT-P 1.97 1.44 96.50 0.084.55E+04 P6GT-P 1.54 1.81 96.54 0.12 4.51E+04

TABLE 38 SEC-HPLC (SUMMARY OF PEAK AREAS); T = 2 month, 4° C.Formulation % Pre- % Pre- % Main % Un- Total ID peak 1 peak 2 Peak PEGArea P6MT 1.02 1.30 97.58 0.11 5.04E+04 H6MT 1.14 1.17 97.62 0.064.88E+04 P6GT 1.26 1.60 97.01 0.12 5.23E+04 P6MS 1.34 1.29 97.31 0.064.99E+04 P6MTMet 1.26 1.30 97.33 0.11 5.00E+04 P7MT 0.74 1.30 97.89 0.074.38E+04 P7GT 0.77 1.52 97.51 0.21 4.80E+04 P6MGT 1.34 1.28 97.26 0.124.62E+04 P6MGT-P 1.54 1.14 97.21 0.10 4.66E+04 P6MT-P 1.97 1.34 96.600.09 4.65E+04 P6GT-P 1.33 1.52 97.08 0.07 4.79E+04

TABLE 39 SEC-HPLC (SUMMARY OF PEAK AREAS); T = 3 month, 4° C. % Pre- %Pre- % Main % Un- Total Formulation peak 1 peak 2 Peak PEG Area P6MT0.72 1.14 98.14 0.00 4.86E+04 H6MT 0.77 0.99 98.24 0.00 5.07E+04 P6GT0.62 1.35 97.96 0.07 5.58E+04 P6MS 0.77 1.04 98.20 0.00 5.19E+04 P6MTMet0.66 0.96 98.39 0.00 5.13E+04 P7MT 0.55 1.06 98.39 0.00 4.15E+04 P7GT0.53 1.42 97.76 0.29 5.08E+04 P6MGT 0.73 1.05 98.22 0.00 4.41E+04P6MGT-P 0.72 1.07 98.21 0.00 4.51E+04 P6MT-P 1.41 1.22 97.37 0.004.66E+04 P6GT-P 1.09 1.54 97.37 0.00 4.59E+04

TABLE 40 SEC-HPLC (SUMMARY OF PEAK AREAS); T = 1 week, 25° C.Formulation % Pre- % Main % Un- Total ID peak Peak PEG Area P6MT 2.1797.70 0.14 4.64E+04 S4MT 3.79 95.39 0.82 4.15E+04 S5MT 2.42 97.31 0.274.46E+04 S5GT 2.35 97.40 0.25 4.59E+04 H6MT 2.10 97.75 0.15 4.63E+04P6GT 2.31 97.54 0.15 5.01E+04 P6MA 1.95 97.92 0.13 4.97E+04 P6MS 2.0697.82 0.12 4.72E+04 P6MTMet 2.14 97.71 0.15 4.85E+04 P7MT 1.79 98.010.19 4.37E+04 P7GT 2.15 97.56 0.30 4.62E+04 P6MGT 2.09 97.75 0.164.17E+04 P6MGT-P 2.19 97.70 0.11 4.33E+04 P6MT-P 2.51 97.41 0.084.23E+04 P6GT-P 2.60 97.21 0.19 4.20E+04

TABLE 41 SEC-HPLC (SUMMARY OF PEAK AREAS); T = 2 week, 25° C.Formulation % Pre- % Main % Un- Total ID peak Peak PEG Area P6MT 1.8798.01 0.12 4.71E+04 S4MT 2.89 96.47 0.65 4.21E+04 S5MT 1.94 97.82 0.244.73E+04 S5GT 2.34 97.43 0.22 4.70E+04 H6MT 2.20 97.66 0.13 4.35E+04P6GT 2.36 97.52 0.12 5.31E+04 P6MA 1.90 98.00 0.10 5.21E+04 P6MS 1.7698.12 0.11 5.07E+04 P6MTMet 1.62 98.30 0.09 5.17E+04 P7MT 2.48 97.370.15 4.28E+04 P7GT 2.08 97.78 0.14 4.94E+04 P6MGT 2.22 97.66 0.124.38E+04 P6MGT-P 1.81 98.06 0.13 4.76E+04 P6MT-P 2.04 97.85 0.114.56E+04 P6GT-P 2.30 97.60 0.10 4.75E+04

TABLE 42 SEC-HPLC (SUMMARY OF PEAK AREAS); T = 4 week, 25° C. % Pre- %Pre- % Main % Un- Total Formulation peak 1 peak 2 Peak PEG Area P6MT1.50 2.31 95.91 0.27 4.37E+04 S4MT 1.25 3.17 94.82 0.76 4.15E+04 S5MT2.47 3.80 93.42 0.32 4.70E+04 S5GT 1.29 2.77 95.57 0.36 4.64E+04 H6MT2.09 1.42 96.25 0.23 4.57E+04 P6GT 1.10 2.29 96.39 0.21 4.79E+04 P6MA1.19 2.48 96.17 0.17 4.72E+04 P6MS 1.60 2.40 95.82 0.19 4.81E+04 P6MTMet1.00 1.65 97.26 0.09 4.87E+04 P7MT 0.92 1.63 97.28 0.17 4.19E+04 P7GT1.02 2.46 96.18 0.34 4.50E+04 P6MGT 1.22 2.16 96.28 0.34 4.08E+04P6MGT-P 1.80 1.81 96.19 0.20 4.21E+04 P6MT-P 1.67 1.96 96.18 0.184.59E+04 P6GT-P 1.45 2.35 95.96 0.24 4.66E+04

TABLE 43 SEC-HPLC (SUMMARY OF PEAK AREAS); T = 6 week, 25° C.Formulation % Pre- % Pre- % Main % Un- Total ID peak 1 peak 2 Peak PEGArea P6MT 0.95 1.91 96.96 0.17 4.68E+04 S5MT 1.32 2.56 95.87 0.254.31E+04 S5GT 1.54 2.96 95.47 0.03 4.64E+04 H6MT 1.74 1.52 96.61 0.124.76E+04 P6GT 1.12 2.24 96.53 0.11 4.80E+04 P6MS 1.41 2.13 96.38 0.084.89E+04 P6MTMet 1.08 1.91 96.85 0.16 4.80E+04 P7MT 0.88 1.82 97.19 0.124.37E+04 P7GT 0.83 2.15 96.91 0.10 4.68E+04 P6MGT 1.25 2.77 95.76 0.214.37E+04 P6MGT-P 1.44 2.30 96.13 0.12 4.37E+04 P6MT-P 2.00 2.08 95.830.08 4.44E+04 P6GT-P 1.62 2.64 95.63 0.11 4.52E+04

TABLE 44 SEC-HPLC (SUMMARY OF PEAK AREAS); T = 2 month, 25° C.Formulation % Pre- % Pre- % Main % Un- Total ID peak 1 peak 2 Peak PEGArea P6MT 1.38 2.16 96.28 0.18 4.85E+04 H6MT 1.56 1.50 96.69 0.255.07E+04 P6GT 1.30 2.61 95.92 0.17 5.22E+04 P6MS 1.36 2.04 96.51 0.095.04E+04 P6MTMet 1.16 1.76 96.98 0.10 5.05E+04 P7MT 0.79 1.81 97.27 0.134.29E+04 P7GT 0.63 2.58 96.40 0.39 4.99E+04 P6MGT 1.05 2.55 96.21 0.194.57E+04 P6MGT-P 1.39 2.22 96.22 0.16 4.56E+04 P6MT-P 1.69 1.96 96.240.12 4.69E+04 P6GT-P 1.42 2.57 95.87 0.14 4.69E+04

TABLE 45 SEC-HPLC (SUMMARY OF PEAK AREAS); T = 3 month, 25° C. % Pre- %Pre- % Main % Un- Total Formulation peak 1 peak 2 Peak PEG Area P6MT0.75 1.88 97.38 0.00 4.89E+04 H6MT 0.86 1.15 97.99 0.00 5.40E+04 P6GT0.92 2.16 96.92 0.00 5.14E+04 P6MS 1.09 2.09 96.71 0.11 4.72E+04 P6MTMet0.92 1.80 97.28 0.00 4.99E+04 P7MT 0.60 1.53 97.87 0.00 4.47E+04 P7GT0.62 2.03 97.17 0.17 4.86E+04 P6MGT 0.86 2.27 96.66 0.20 4.49E+04P6MGT-P 0.93 2.04 96.85 0.18 4.48E+04 P6MT-P 1.03 1.76 97.21 0.004.72E+04 P6GT-P 1.05 2.17 96.63 0.15 4.68E+04

TABLE 46 SEC-HPLC (SUMMARY OF PEAK AREAS); T = 1 week, 40° C.Formulation % Pre- % Main % Un- Total ID peak Peak PEG Area P6MT 2.3297.51 0.17 4.61E+04 S4MT 7.69 91.40 0.91 4.01E+04 S5MT 3.01 96.68 0.304.69E+04 S5GT 3.18 96.52 0.30 4.36E+04 H6MT 2.44 97.34 0.22 4.80E+04P6GT 2.67 97.17 0.16 5.01E+04 P6MA 5.24 94.52 0.24 4.77E+04 P6MS 3.6996.13 0.18 4.84E+04 P6MTMet 2.33 97.44 0.23 4.55E+04 P7MT 2.60 97.240.16 4.31E+04 P7GT 2.59 97.18 0.23 4.61E+04 P6MGT 2.85 96.96 0.194.58E+04 P6MGT-P 3.20 96.69 0.12 4.24E+04 P6MT-P 3.42 96.44 0.133.95E+04 P6GT-P 3.30 96.57 0.13 4.27E+04

TABLE 47 SEC-HPLC (SUMMARY OF PEAK AREAS); T = 2 week, 40° C.Formulation % Pre- % Main % Un- Total ID peak Peak PEG Area P6MT 2.4597.45 0.11 4.54E+04 S4MT 7.29 91.94 0.77 4.29E+04 S5MT 4.22 95.48 0.304.67E+04 S5GT 3.63 96.18 0.19 4.65E+04 H6MT 2.41 97.36 0.22 4.72E+04P6GT 2.82 97.05 0.13 5.09E+04 P6MA 41.10 58.76 0.14 4.92E+04 P6MS 4.1595.71 0.14 5.14E+04 P6MTMet 2.29 97.53 0.18 4.96E+04 P7MT 2.07 97.870.06 4.43E+04 P7GT 2.15 97.74 0.12 5.06E+04 P6MGT 3.29 96.53 0.194.40E+04 P6MGT-P 2.92 97.00 0.07 4.80E+04 P6MT-P 2.65 97.23 0.124.68E+04 P6GT-P 2.50 97.40 0.10 4.92E+04

TABLE 48 SEC-HPLC (SUMMARY OF PEAK AREAS); T = 4 week, 40° C.Formulation % Pre- % Pre- % Main % Un- Total ID peak 1 peak 2 Peak PEGArea P6MT 1.22 3.47 94.87 0.44 4.55E+04 S4MT 0.00 12.63 86.23 1.144.06E+04 S5MT 1.43 4.82 93.21 0.54 4.53E+04 S5GT 1.29 4.54 93.79 0.384.48E+04 H6MT 1.55 2.05 95.93 0.47 4.45E+04 P6GT 1.21 4.14 94.37 0.284.90E+04 P6MA 0.00 63.87 35.95 0.18 4.75E+04 P6MS 0.00 8.35 91.27 0.374.71E+04 P6MTMet 1.32 3.60 94.35 0.73 4.59E+04 P7MT 0.80 2.18 96.89 0.124.21E+04 P7GT 0.75 2.81 96.13 0.31 4.60E+04 P6MGT 1.42 4.38 93.85 0.363.89E+04 P6MGT-P 1.36 3.46 94.93 0.25 4.23E+04 P6MT-P 1.69 3.52 94.470.32 4.04E+04 P6GT-P 1.45 3.60 94.70 0.25 4.50E+04

TABLE 49 SEC-HPLC (SUMMARY OF PEAK AREAS); T = 6 week, 40° C.Formulation % Pre- % Pre- % Main % % Un- Total ID peak 1 peak 2 PeakClip PEG Area P6MT 1.13 3.12 95.54 0.00 0.21 4.52E+04 S5MT 1.68 6.5991.30 0.00 0.42 4.34E+04 S5GT 1.05 4.82 93.78 0.00 0.34 4.48E+04 H6MT1.36 2.16 96.12 0.00 0.35 4.61E+04 P6GT 0.88 3.38 95.65 0.00 0.104.93E+04 P6MS 0.00 10.02 88.34 1.31 0.33 4.82E+04 P6MTMet 1.24 3.3593.80 1.16 0.46 4.98E+04 P7MT 1.03 3.38 95.35 0.00 0.25 4.15E+04 P7GT0.77 3.19 95.86 0.00 0.19 4.73E+04 P6MGT 1.12 4.92 93.58 0.00 0.394.08E+04 P6MGT-P 1.35 4.23 94.16 0.00 0.25 4.46E+04 P6MT-P 1.80 3.7094.29 0.00 0.21 4.51E+04 P6GT-P 1.41 3.81 94.59 0.00 0.19 4.56E+04

TABLE 50 SEC-HPLC (SUMMARY OF PEAK AREAS); T = 2 month, 40° C. % Pre- %Pre- % Main % Un- Total Formulation peak 1 peak 2 Peak PEG Area P6MT1.15 3.04 95.58 0.23 4.81E+04 H6MT 1.34 2.07 96.32 0.27 5.15E+04 P6GT1.15 3.46 95.22 0.17 5.24E+04 P6MS 0.00 10.51 89.14 0.36 5.08E+04P6MTMet 1.24 4.73 93.27 0.76 4.89E+04 P7MT 0.71 2.74 96.30 0.25 4.33E+04P7GT 0.66 3.71 95.25 0.37 4.78E+04 P6MGT 0.93 4.25 94.53 0.28 4.60E+04P6MGT-P 1.23 4.03 94.48 0.25 4.58E+04 P6MT-P 1.51 3.67 94.57 0.254.71E+04 P6GT-P 1.20 4.06 94.55 0.19 4.72E+04

RP-HPLC: FIG. 21 is an example RP-HPLC integration. Tables 51-68 showRP-HPLC results from this study.

TABLE 51 RP-HPLC (SUMMARY OF PEAK AREAS); T = 0 Formulation % Pre- %Post- % Main % Un- Total ID peak peak Peak PEG Area P6MT 0.78 1.09 98.020.11 5.78E+03 S4MT 0.83 2.42 96.53 0.22 5.36E+03 S5MT 0.69 1.08 98.34−0.11 5.92E+03 S5GT 0.83 1.57 97.52 0.08 6.26E+03 H6MT 0.93 0.95 98.060.06 6.57E+03 P6GT 0.73 0.98 98.21 0.07 6.75E+03 P6MA 1.00 0.70 98.38−0.08 6.32E+03 P6MS 0.92 0.77 98.30 0.01 6.74E+03 P6MTMet 0.93 0.8198.27 −0.01 6.49E+03 P7MT 1.12 0.77 98.11 0.01 6.64E+03 P7GT 0.95 0.8198.20 0.04 6.12E+03 P6MGT 1.11 1.04 97.77 0.09 5.56E+03 P6MGT-P 0.980.88 98.29 −0.15 5.48E+03 P6MT-P 1.18 0.83 98.07 −0.08 5.63E+03 P6GT-P0.89 1.05 98.10 −0.04 4.72E+03

TABLE 52 RP-HPLC (SUMMARY OF PEAK AREAS); T = 1 week: 4° C. Formulation% Pre- % Pre- % Post- % Main % Un- Total ID peak 1 peak 2 peak Peak PEGArea P6MT 0.30 1.15 1.47 96.90 0.19 7.38E+03 S4MT 0.31 1.26 2.55 95.410.46 6.76E+03 S5MT 0.54 1.37 1.72 96.18 0.19 6.47E+03 S5GT 0.26 1.321.73 96.45 0.24 6.40E+03 H6MT 0.28 1.23 1.05 97.38 0.06 6.51E+03 P6GT0.29 1.32 1.50 96.64 0.25 7.22E+03 P6MA 0.37 1.36 1.02 97.19 0.067.22E+03 P6MS 0.22 1.03 1.09 97.57 0.09 7.12E+03 P6MTMet 0.25 1.14 1.1697.28 0.17 7.07E+03 P7MT 0.26 1.11 1.13 97.34 0.17 7.05E+03 P7GT 0.282.31 1.28 95.98 0.14 6.71E+03 P6MGT 0.43 1.46 1.44 96.32 0.35 7.08E+03P6MGT-P 0.39 1.19 1.43 96.89 0.10 6.21E+03 P6MT-P 0.38 1.27 1.08 97.020.25 7.70E+03 P6GT-P 0.47 1.39 1.30 96.47 0.37 7.64E+03

TABLE 53 RP-HPLC (SUMMARY OF PEAK AREAS): T = 2 week; 4° C. Formulation% Pre- % Pre- % Post- % Main % Un- Total ID peak 1 peak 2 peak Peak PEGArea P6MT 0.27 0.85 0.78 98.05 0.04 7.17E+03 S4MT 0.29 1.09 2.48 95.590.55 5.75E+03 S5MT 0.14 1.16 1.45 97.25 −0.01 5.57E+03 S5GT 0.21 1.291.46 96.99 0.04 6.41E+03 H6MT 0.34 1.14 0.99 97.50 0.03 6.34E+03 P6GT0.27 1.26 1.33 97.02 0.12 6.78E+03 P6MA 0.33 1.17 0.87 97.70 −0.077.96E+03 P6MS 0.42 1.31 0.84 97.43 −0.01 7.16E+03 P6MTMet 0.77 1.37 0.9096.99 −0.03 7.32E+03 P7MT 0.27 1.32 0.66 97.83 −0.08 6.08E+03 P7GT 0.451.40 1.23 96.85 0.06 6.67E+03 P6MGT 0.41 1.35 0.92 97.32 0.00 6.20E+03P6MGT-P 0.38 1.42 0.88 97.35 −0.03 6.34E+03 P6MT-P 0.48 1.29 0.90 97.36−0.03 6.83E+03 P6GT-P 0.40 1.64 1.23 96.68 0.06 6.18E+03

TABLE 54 RP-HPLC (SUMMARY OF PEAK AREAS); T = 4 week; 4° C. Formulation% Pre- % Pre- % Post- % Main % Un- Total ID peak 1 peak 2 peak Peak PEGArea P6MT 0.19 0.95 1.03 97.71 0.12 6.82E+03 S4MT 0.23 1.06 2.57 95.560.58 5.80E+03 S5MT 0.21 1.15 1.56 96.97 0.11 7.10E+03 S5GT 0.18 1.412.08 96.09 0.24 5.23E+03 H6MT 0.11 1.07 0.88 97.82 0.13 7.73E+03 P6GT0.14 1.17 1.14 97.41 0.14 7.40E+03 P6MA 0.20 1.01 0.98 97.72 0.107.18E+03 P6MS 0.14 0.90 1.06 97.78 0.13 7.38E+03 P6MTMet 0.17 0.79 0.9997.97 0.09 7.86E+03 P7MT 0.05 1.00 1.12 97.83 0.00 6.25E+03 P7GT 0.150.87 1.38 97.44 0.17 7.06E+03 P6MGT −0.04 0.94 0.81 98.19 0.10 5.72E+03P6MGT-P 0.17 1.07 1.11 97.60 0.05 6.30E+03 P6MT-P 0.08 1.07 0.96 97.90−0.02 6.66E+03 P6GT-P 0.08 1.17 1.39 97.25 0.12 6.33E+03

TABLE 55 RP-HPLC (SUMMARY OF PEAK AREAS); T = 6 week; 4° C. Formulation% Pre- % Pre- % Post- % Main % Un- Total ID peak 1 peak 2 peak Peak PEGArea P6MT 0.09 1.19 1.68 96.95 0.09 6.40E+03 S5MT 0.08 1.27 2.12 96.280.26 6.09E+03 S5GT 0.18 1.47 2.38 95.74 0.22 6.67E+03 H6MT 0.14 1.081.55 97.15 0.07 6.73E+03 P6GT 0.26 1.34 1.83 96.32 0.25 6.76E+03 P6MS0.26 1.10 1.36 97.25 0.03 7.26E+03 P6MTMet 0.14 1.15 1.40 97.29 0.027.34E+03 P7MT 0.28 1.29 1.60 96.73 0.11 6.21E+03 P7GT 0.36 1.21 1.8196.33 0.29 6.67E+03 P6MGT 0.46 1.44 1.83 96.26 0.00 5.56E+03 P6MGT-P0.30 1.19 1.43 97.05 0.03 6.41E+03 P6MT-P 0.13 1.10 1.22 97.58 −0.036.41E+03 P6GT-P 0.16 1.04 1.37 97.32 0.11 6.28E+03

TABLE 56 RP-HPLC (SUMMARY OF PEAK AREAS); T = 2 month; 4° C. Formulation% Pre- % Pre- % Post- % Main % Un- Total ID peak 1 peak 2 peak Peak PEGArea P6MT 0.10 0.79 1.39 97.54 0.18 6.73E+03 H6MT 0.31 0.80 1.34 97.370.17 6.50E+03 P6GT 0.37 0.97 1.48 96.94 0.24 7.49E+03 P6MS 0.45 0.691.28 97.43 0.15 6.90E+03 P6MTMet 0.20 0.74 1.23 97.67 0.17 7.07E+03 P7MT0.32 0.76 1.26 97.61 0.06 5.84E+03 P7GT 0.38 0.77 1.68 96.79 0.387.10E+03 P6MGT 0.51 0.85 1.32 97.02 0.30 5.87E+03 P6MGT-P 0.17 0.96 1.4197.19 0.27 6.46E+03 P6MT-P 0.13 0.90 1.52 97.37 0.09 6.20E+03 P6GT-P0.18 0.91 1.59 97.09 0.23 6.56E+03

TABLE 57 RP-HPLC (SUMMARY OF PEAK AREAS); T = 3 month; 4° C. Formulation% Pre- % Pre- % Post- % Main % Un- Total ID peak 1 peak 2 peak Peak PEGArea P6MT 0.30 1.06 1.44 96.79 0.41 7.70E+03 H6MT 0.38 1.08 1.47 96.790.27 7.70E+03 P6GT 0.39 1.24 1.57 96.43 0.37 9.07E+03 P6MS 0.45 1.291.29 96.90 0.07 8.43E+03 P6MTMet 0.23 1.11 1.30 97.22 0.14 8.48E+03 P7MT0.29 1.13 1.51 96.96 0.11 7.25E+03 P7GT 0.45 1.26 2.04 95.83 0.428.53E+03 P6MGT 0.61 1.45 1.93 95.55 0.45 7.68E+03 P6MGT-P 0.19 1.24 1.5896.74 0.26 7.71E+03 P6MT-P 0.25 1.04 1.50 97.08 0.14 7.80E+03 P6GT-P0.33 1.22 1.79 96.32 0.35 7.83E+03

TABLE 58 RP-HPLC (SUMMARY OF PEAK AREAS); T = 1 week; 25° C. Formulation% Pre- % Pre- % Post- % Main % Un- Total ID peak 1 peak 2 peak Peak PEGArea P6MT 0.46 1.41 1.51 96.48 0.14 7.80E+03 S4MT 0.60 1.45 3.15 94.130.66 6.90E+03 S5MT 0.52 1.49 1.98 95.77 0.24 6.64E+03 S5GT 0.48 1.681.93 95.70 0.21 6.45E+03 H6MT 0.50 1.39 1.33 96.59 0.19 8.69E+03 P6GT0.50 1.82 1.76 95.63 0.30 7.78E+03 P6MA 0.61 1.43 1.11 96.76 0.098.95E+03 P6MS 0.60 1.44 1.32 96.59 0.05 7.14E+03 P6MTMet 0.37 1.34 1.4196.81 0.06 6.83E+03 P7MT 0.53 1.38 1.28 96.77 0.03 6.52E+03 P7GT 0.631.60 1.78 95.69 0.30 7.06E+03 P6MGT 0.63 1.84 1.46 95.90 0.16 6.14E+03P6MGT-P 0.77 1.74 1.60 95.71 0.19 6.68E+03 P6MT-P 0.70 1.55 1.35 96.340.07 7.01E+03 P6GT-P 0.64 1.92 1.73 95.42 0.29 6.68E+03

TABLE 59 RP-HPLC (SUMMARY OF PEAK AREAS); T = 2 week; 25° C. Formulation% Pre- % Pre- % Post- % Main % Un- Total ID peak 1 peak 2 peak Peak PEGArea P6MT 0.41 1.39 1.35 96.77 0.08 6.97E+03 S4MT 0.35 1.35 3.06 94.750.48 5.69E+03 S5MT 0.44 1.38 2.08 96.02 0.08 6.96E+03 S5GT 1.48 3.744.43 90.02 0.32 3.34E+03 H6MT 0.58 1.38 1.32 96.54 0.17 7.68E+03 P6GT0.52 1.82 1.84 95.57 0.26 7.72E+03 P6MA 0.44 1.31 0.85 97.44 −0.057.58E+03 P6MS 0.46 1.39 1.02 97.13 0.00 7.65E+03 P6MTMet 0.43 1.44 1.3996.59 0.15 7.00E+03 P7MT 0.47 1.33 1.13 97.14 −0.07 6.10E+03 P7GT 0.641.80 2.24 94.87 0.44 7.21E+03 P6MGT 0.58 2.03 1.42 95.83 0.14 6.94E+03P6MGT-P 0.53 1.94 1.23 96.22 0.07 6.55E+03 P6MT-P 0.50 1.39 1.27 96.780.05 6.98E+03 P6GT-P 0.54 1.82 1.38 96.25 0.01 6.60E+03

TABLE 60 RP-HPLC (SUMMARY OF PEAK AREAS); T = 4 week; 25° C. Formulation% Pre- % Pre- % Post- % Main % Un- Total ID peak 1 peak 2 peak Peak PEGArea P6MT 0.18 1.08 2.33 96.16 0.26 6.68E+03 S4MT 0.39 1.27 3.61 94.060.67 5.94E+03 S5MT 0.19 1.09 2.98 95.49 0.25 6.68E+03 S5GT 0.20 1.553.20 94.70 0.35 6.31E+03 H6MT 0.28 0.91 1.59 96.95 0.28 6.37E+03 P6GT0.30 1.88 2.40 95.10 0.32 7.03E+03 P6MA 0.16 1.37 1.79 96.57 0.116.95E+03 P6MS 0.22 1.22 1.52 96.95 0.09 7.06E+03 P6MTMet 0.19 1.09 1.2597.40 0.07 7.21E+03 P7MT 0.22 1.06 1.31 97.35 0.06 6.68E+03 P7GT 0.441.42 2.07 95.37 0.70 6.47E+03 P6MGT 0.49 1.75 1.49 95.99 0.28 5.50E+03P6MGT-P 0.42 2.04 1.31 96.14 0.08 6.06E+03 P6MT-P 0.38 0.90 1.33 97.280.12 6.70E+03 P6GT-P 0.35 1.75 1.76 95.90 0.24 7.52E+03

TABLE 61 RP-HPLC (SUMMARY OF PEAK AREAS); T = 6 week; 25° C. Formulation% Pre- % Pre- % Post- % Main % Un- Total ID peak 1 peak 2 peak Peak PEGArea P6MT 0.26 0.99 1.47 97.05 0.23 6.36E+03 S5MT 0.40 1.05 2.47 95.910.17 6.09E+03 S5GT 0.42 1.85 2.96 94.46 0.31 6.52E+03 H6MT 0.48 0.861.65 96.87 0.14 5.97E+03 P6GT 0.25 1.57 1.65 96.38 0.15 7.14E+03 P6MS0.35 1.02 1.08 97.58 −0.03 7.07E+03 P6MTMet 0.20 1.05 1.73 96.81 0.216.93E+03 P7MT −0.01 0.91 1.27 97.90 −0.07 6.09E+03 P7GT 0.10 1.08 1.2897.58 −0.05 6.66E+03 P6MGT 0.32 2.28 2.25 94.95 0.20 6.49E+03 P6MGT-P0.26 2.03 1.79 95.77 0.15 6.82E+03 P6MT-P 0.08 0.95 1.33 97.67 −0.035.91E+03 P6GT-P 0.43 2.52 1.79 95.17 0.09 6.78E+03

TABLE 62 RP-HPLC (SUMMARY OF PEAK AREAS); T = 2 month; 25° C. % Pre- %Pre- % Post- % Main % Un- Total Formulation peak 1 peak 2 peak Peak PEGArea P6MT 0.44 1.13 2.42 95.52 0.49 7.00E+03 H6MT 0.54 0.84 2.35 95.380.90 7.53E+03 P6GT 0.52 1.73 2.39 94.84 0.52 6.99E+03 P6MS 0.27 1.141.46 96.91 0.21 7.02E+03 P6MTMet 0.18 1.25 1.81 96.43 0.34 6.96E+03 P7MT0.38 1.06 1.93 96.39 0.24 6.10E+03 P7GT 0.64 1.76 3.07 93.62 0.917.35E+03 P6MGT 0.52 2.41 2.62 93.97 0.49 6.79E+03 P6MGT-P 0.46 2.26 2.5494.33 0.41 6.56E+03 P6MT-P 0.37 1.28 1.98 96.12 0.25 7.89E+03 P6GT-P0.51 2.04 2.69 94.21 0.54 6.63E+03

TABLE 63 RP-HPLC (SUMMARY OF PEAK AREAS); T = 3 month; 25° C.Formulation % Pre- % Pre- % Post- % Main % Un- Total ID peak 1 peak 2peak Peak PEG Area P6MT 0.41 1.31 2.57 95.23 0.48 8.23E+03 H6MT 0.431.18 1.86 96.24 0.29 8.73E+03 P6GT 0.42 1.82 2.27 95.02 0.47 8.61E+03P6MS 0.47 1.23 1.98 96.09 0.24 8.22E+03 P6MTMet 0.28 1.19 2.84 95.060.63 8.13E+03 P7MT 0.36 1.03 1.74 96.70 0.16 7.47E+03 P7GT 0.39 1.701.77 95.77 0.38 8.46E+03 P6MGT 0.34 2.14 2.72 94.16 0.64 7.47E+03P6MGT-P 0.35 2.28 2.53 94.26 0.58 7.58E+03 P6MT-P 0.27 1.28 1.89 96.380.18 7.62E+03 P6GT-P 0.36 1.70 2.56 94.98 0.40 7.95E+03

TABLE 64 RP-HPLC (SUMMARY OF PEAK AREAS): T = 1 week: 40° C. Formulation% Pre- % Pre- % Post- % Main % Un- Total ID peak 1 peak 2 peak Peak PEGArea P6MT 0.60 1.43 2.12 95.68 0.18 7.13E+03 S4MT 0.71 1.92 6.01 90.690.66 6.11E+03 S5MT 0.60 1.60 3.25 94.35 0.19 7.72E+03 S5GT 0.76 2.203.13 93.73 0.19 6.77E+03 H6MT 0.74 1.39 1.98 95.58 0.30 7.61E+03 P6GT0.81 2.13 2.27 94.44 0.35 7.50E+03 P6MA 0.68 1.60 1.48 96.04 0.208.34E+03 P6MS 0.84 1.59 2.22 95.02 0.32 8.12E+03 P6MTMet 0.74 1.50 2.1695.27 0.34 8.28E+03 P7MT 0.63 1.42 1.91 95.88 0.16 6.56E+03 P7GT 0.541.98 2.34 94.80 0.35 7.81E+03 P6MGT 0.70 2.41 2.19 94.30 0.41 7.73E+03P6MGT-P 0.44 2.12 1.80 95.52 0.13 7.65E+03 P6MT-P 0.58 1.37 2.18 95.650.21 6.83E+03 P6GT-P 0.40 2.00 2.21 95.17 0.23 7.48E+03

TABLE 65 RP-HPLC (SUMMARY OF PEAK AREAS); T = 2 week; 40° C. Formulation% Pre- % Pre- % Post- % Main % Un- Total ID peak 1 peak 2 peak Peak PEGArea P6MT 0.59 1.58 2.44 95.12 0.27 7.33E+03 S4MT 0.46 2.13 8.25 88.500.66 5.73E+03 S5MT 0.46 1.54 4.45 93.42 0.12 6.88E+03 S5GT 0.44 2.303.92 93.07 0.28 6.73E+03 H6MT 0.70 1.28 2.54 94.85 0.63 7.06E+03 P6GT0.56 2.02 2.43 94.80 0.20 6.86E+03 P6MA 0.55 1.58 6.98 90.67 0.227.78E+03 P6MS 0.47 1.39 2.27 95.63 0.24 7.56E+03 P6MTMet 0.29 1.39 2.7195.34 0.27 6.20E+03 P7MT 0.34 1.12 1.83 96.65 0.07 6.91E+03 P7GT 0.361.55 1.80 96.20 0.09 7.28E+03 P6MGT 0.30 2.01 2.39 95.08 0.22 6.56E+03P6MGT-P 0.35 2.11 2.49 94.91 0.13 6.70E+03 P6MT-P 0.38 1.23 2.37 95.900.12 7.00E+03 P6GT-P 0.49 1.96 2.58 94.83 0.14 7.03E+03

TABLE 66 RP-HPLC (SUMMARY OF PEAK AREAS); T = 4 week; 40° C. Formulation% Pre- % Pre- % Post- % Main % Un- Total ID peak 1 peak 2 peak Peak PEGArea P6MT 0.47 1.47 4.21 93.29 0.56 6.70E+03 S4MT 0.53 2.24 14.40 81.741.09 6.61E+03 S5MT 0.67 1.69 7.09 90.13 0.43 6.72E+03 S5GT 0.52 2.515.89 90.79 0.29 6.35E+03 H6MT 0.79 1.38 3.09 94.01 0.73 6.38E+03 P6GT0.61 2.48 3.77 92.71 0.42 7.72E+03 P6MA 1.09 1.59 23.19 73.63 0.507.25E+03 P6MS 0.81 1.13 3.86 93.78 0.43 7.48E+03 P6MTMet 0.46 1.33 4.7092.35 1.17 6.66E+03 P7MT 0.38 1.31 2.12 96.12 0.08 6.18E+03 P7GT 0.291.89 2.78 94.68 0.36 7.33E+03 P6MGT 0.32 1.97 4.59 92.73 0.38 6.82E+03P6MGT-P 0.31 1.90 3.37 94.24 0.19 6.39E+03 P6MT-P 0.41 1.43 3.83 94.040.28 5.63E+03 P6GT-P 0.35 2.11 3.79 93.46 0.29 6.38E+03

TABLE 67 RP-HPLC (SUMMARY OF PEAK AREAS): T = 6 week: 40° C. Formulation% Pre- % Pre- % Post- % Main % Un- Total ID peak 1 peak 2 peak Peak PEGArea P6MT 0.48 1.58 3.29 94.59 0.07 6.70E+03 S5MT 0.27 2.23 9.81 87.580.11 6.66E+03 S5GT 0.15 2.99 6.37 90.33 0.16 6.71E+03 H6MT 0.33 1.362.71 95.19 0.41 6.64E+03 P6GT 0.11 2.43 3.30 94.12 0.05 7.39E+03 P6MS0.90 2.67 6.46 89.44 0.53 6.93E+03 P6MTMet 0.00 1.59 2.87 94.76 0.787.26E+03 P7MT 0.32 1.26 3.09 95.11 0.21 5.32E+03 P7GT 0.29 2.12 2.3894.97 0.23 6.63E+03 P6MGT 0.31 2.56 5.93 91.06 0.14 6.51E+03 P6MGT-P0.22 2.62 4.53 92.48 0.16 6.75E+03 P6MT-P 0.23 1.83 3.76 94.03 0.156.93E+03 P6GT-P 0.17 2.65 3.06 93.99 0.12 7.11E+03

TABLE 68 RP-HPLC (SUMMARY OF PEAK AREAS); T = 2 month; 40° C.Formulation % Pre- % Pre- % Post- % Main % Un- Total ID peak 1 peak 2peak Peak PEG Area P6MT 0.57 1.69 4.69 92.50 0.55 7.07E+03 H6MT 0.551.46 3.78 93.40 0.81 7.15E+03 P6GT 0.29 2.41 3.89 93.14 0.27 7.77E+03P6MS 1.16 2.25 7.34 88.30 0.96 6.88E+03 P6MTMet 0.51 2.17 8.43 86.142.75 7.32E+03 P7MT 0.53 1.39 3.69 93.98 0.41 6.55E+03 P7GT 0.58 2.324.06 92.38 0.66 7.03E+03 P6MGT 0.56 2.39 6.49 90.06 0.50 7.32E+03P6MGT-P 0.49 2.30 5.63 91.06 0.51 6.77E+03 P6MT-P 0.64 1.87 5.61 91.270.61 7.28E+03 P6GT-P 0.38 2.64 4.54 91.99 0.43 7.06E+03

CEX/IEX analysis from this study is shown in Tables 69-71.

TABLE 69 IEX-HPLC (SUMMARY OF PEAK AREAS); T = 0 Formulation % Pre- %Main Post- Total ID peak Peak peak Area P6MT 6.27 91.01 2.72 1343 S4MT2.95 93.19 3.86 991 S5MT 2.11 94.21 3.67 997 S5GT 2.19 94.46 3.35 1074H6MT 1.92 95.63 2.46 1007 P6GT 4.55 93.11 2.34 1140 P6MA 4.80 94.17 1.031172 P6MS 3.67 95.03 1.31 1158 P6MTMet 4.47 94.04 1.49 987 P7MT 0.6198.84 0.55 876 P7GT 0.94 96.39 2.67 993 P6MGT 1.62 95.31 3.07 959P6MGT-P 1.68 96.16 2.15 920 P6MT-P 1.56 96.32 2.12 926 P6GT-P 1.89 94.833.28 1005

TABLE 70 IEX-HPLC (SUMMARY OF PEAK AREAS); T = 1 week; 40° C.Formulation % Pre- % Main Post- Total ID peak Peak peak Area P6MT 2.2293.02 4.76 1.96E+03 S4MT 3.88 90.09 6.03 1.41E+03 S5MT 2.79 91.88 5.341.88E+03 S5GT 2.80 92.21 4.99 1.82E+03 H6MT 3.53 92.54 3.93 1.35E+03P6GT 2.87 92.89 4.24 2.12E+03 P6MA 0.00 95.97 4.03 7.14E+02 P6MS 4.3491.68 3.98 2.01E+03 P6MTMet 2.24 93.82 3.94 1.95E+03 P7MT 6.21 91.272.52 1.86E+03 P7GT 4.38 86.25 9.37 2.01E+03 P6MGT 1.25 96.69 2.061.60E+03 P6MGT-P 1.57 95.53 2.89 1.82E+03 P6MT-P 2.55 94.83 2.621.70E+03 P6GT-P 2.68 93.80 3.53 1.80E+03

TABLE 71 IEX-HPLC (SUMMARY OF PEAK AREAS): T = 2 week: 40° C.Formulation % Pre- % Pre- % Main Post- Total ID peak 1 peak 2 Peak peakArea P6MT 0.00 5.28 89.24 5.48 1920.32 S4MT 3.16 8.27 79.70 8.87 1510.4S5MT 1.46 3.52 88.48 6.54 1706.32 S5GT 1.65 3.63 88.79 5.93 1767.78 H6MT0.00 4.04 90.95 5.02 1977.59 P6GT 0.00 3.13 91.63 5.24 2029.88 P6MA 0.005.18 85.28 9.54 2016.34 P6MS 0.00 3.60 90.91 5.49 1954 P6MTMet 0.00 5.2192.41 2.38 1942.53 P7MT 0.00 3.10 93.61 3.29 1668.32 P7GT 0.00 3.4891.71 4.80 1987.09 P6MGT 0.00 3.05 92.98 3.98 1703.47 P6MGT-P 0.00 2.8992.00 5.12 1812.89 P6MT-P 0.00 4.49 90.40 5.12 1818.52 P6GT-P 0.00 3.7691.13 5.10 1893.99

Lower pH formulations were studied for 4 weeks (pH 4) and 6 weeks (pH5). For the time-frame studied, the following trends were found.SEC-HPLC and RP-HPLC analysis showed that lower pH formulations (pH 4and 5) generated increased levels of unPEGylated material after storageof lyophilized material at 4° C. than higher pH formulations (pH 6 and7). In addition, higher aggregate levels were observed at lower pHformulations at 25 and 40° C. than with the pH 6 and 7 formulations.Also, pH 6 and 7 formulations exhibited a greater % of main peak areathan the lower pH formulations as analyzed by cIEX-HPLC.

Throughout the 3 month study timeframe with storage of lyophilized hGHat 25 and 40° C., formulations with Histidine showed the least amount ofhigh molecular weight aggregates by SDS-PAGE. Formulations containingmannitol in combination with glycine were found to be stabilized hGHagainst agitation induced aggregation, showing that the combination is agood bulking agent. However, histidine in combination with mannitol alsodemonstrated stabilization of hGH against agitation induced aggregation.Trehalose was an effective stabilizer.

Example 15 Injection Feasibility Study

Injection feasibility studies were performed using a formulation ofPEGylated hGH at four different concentrations. PEG-hGH in 20 mM sodiumcitrate, 2% glycine, 0.5% mannitol, pH 6 was buffer exchanged into 10 mMNaH₂PO₄, 4% mannitol, 2% trehalose, 0.01% polysorbate 20 (PS20), pH 6.0(Formulation ID=P6MT) via centrifugal concentration. The PEG-hGH in P6MTbuffer was concentrated to 8, 10, 12, and 14 mg/mL. 1 mL fill was placedinto vials, and the samples were lyophilized. Lyophilized vials werereconstituted with 1 mL of water. Injection feasibility was tested bypushing 1 mL of each concentration through 27 gauge needle with 4 lbs offorce. Instant reconstitution was found for all concentrations ofPEGylated hGH tested. 8 mg/mL PEGylated hGH injected in 3.5 s (seconds);10 mg/mL injected in 3.6 s; 12 mg/mL injected in 3.9 s; and 14 mg/mLinjected in 4.5 s.

Additional injection feasibility studies were performed with 30 gaugeneedles. PEG-hGH, in 10 mM NaH₂PO₄, 4% mannitol, 2% trehalose, 0.01%polysorbate 20, pH 6.0 (Formulation ID=P6MT buffer), at 8, 10, 12, and14 mg/mL was tested for injection feasibility with a 30 gauge needle.The samples were pushed through a 30 gauge needle under 8 lbs. of forceand timed for duration. When 1 mL of each concentration was pushedthrough a 30 gauge needle under 8 lbs. of force, the 8 mg/mL, 10 mg/mL,12 mg/mL and 14 mg/mL concentrations injected in 7.4 s (seconds), 9.9 s,9.9 s, and 10.7 s, respectively.

Injection of 1 cc within 10 seconds under 8 lbs. of force is generallyaccepted as the standard for injection since the average person canapply up to 8 lbs. of force on a syringe.

Example 16 One Week Reconstitution Study

For this study, reconstituted samples were stored for one week at 4° C.The formulations matrix used for this study was the same as Table 13.After storage for a week, the concentration and pH were measured. Thesamples were also analyzed by SEC-HPLC, RP-HPLC, and SDS-PAGE as shownin Tables 72-74 and FIGS. 59-60.

TABLE 72 CONCENTRATION, pH 1 week reconstitution 4° C. Formulation Conc.ID (mg/mL) pH P6MT 1.6 6.17 S4MT 1.5 4.05 S5MT 1.6 4.98 S5GT 1.7 5.06H6MT 1.8 6.05 P6GT 1.9 6.10 P6MA 1.9 6.39 P6MS 1.8 6.24 P6MTMet 1.9 6.13P7MT 1.6 6.93 P7GT 1.8 6.94 P6MGT 1.6 6.20 P6MGT-P 1.7 6.16 P6MT-P 1.76.14 P6GT-P 1.7 6.14

TABLE 73 SEC-HPLC (SUMMARY OF PEAK AREAS); 1 week reconstitution 4° C.Formulation % Pre- % Pre- % Main % Un- Total ID peak 1 peak 2 Peak PEGArea P6MT 2.32 1.44 96.06 0.18 4.23E+04 S4MT 0.93 1.88 95.68 1.514.13E+04 S5MT 1.73 1.46 96.38 0.43 4.32E+04 S5GT 1.49 1.62 96.47 0.424.63E+04 H6MT 1.86 1.39 96.57 0.17 4.83E+04 P6GT 1.50 1.49 96.84 0.174.95E+04 P6MA 1.69 1.61 96.50 0.20 5.03E+04 P6MS 1.74 1.43 96.63 0.204.71E+04 P6MTMet 2.09 1.42 96.31 0.18 4.83E+04 P7MT 1.44 1.30 97.16 0.104.16E+04 P7GT 1.61 1.51 96.67 0.21 4.77E+04 P6MGT 1.47 1.24 97.02 0.274.43E+04 P6MGT-P 1.92 1.16 96.69 0.22 4.57E+04 P6MT-P 2.06 1.36 96.380.20 4.67E+04 P6GT-P 2.10 1.53 96.18 0.19 4.55E+04

TABLE 74 RP-HPLC (SUMMARY OF PEAK AREAS); 1 week reconstitution 4° C.Formulation % Pre- % Pre- % Post- % Main % Un- Total ID peak 1 peak 2peak Peak PEG Area P6MT 0.52 1.13 1.76 96.43 0.16 6.16E+03 S4MT 0.331.11 3.27 94.38 0.90 5.96E+03 S5MT 0.27 1.21 1.89 96.49 0.13 7.11E+03S5GT 0.21 1.34 1.91 96.40 0.14 6.49E+03 H6MT 0.31 1.16 1.24 97.17 0.118.40E+03 P6GT 0.18 1.19 1.37 97.04 0.22 8.09E+03 P6MA 0.27 1.15 1.4097.09 0.09 6.15E+03 P6MS 0.29 1.16 1.45 96.95 0.15 6.51E+03 P6MTMet 0.161.02 1.27 97.45 0.10 7.45E+03 P7MT 0.38 1.17 1.11 97.31 0.03 6.47E+03P7GT 0.29 1.26 1.12 97.11 0.21 8.23E+03 P6MGT 0.31 1.32 1.60 96.52 0.265.95E+03 P6MGT-P 0.25 1.23 1.15 97.26 0.10 6.84E+03 P6MT-P 0.24 1.051.31 97.26 0.14 7.22E+03 P6GT-P 0.27 1.27 1.29 96.92 0.26 6.73E+03

One week storage of reconstituted samples at 4° C. resulted in slightincrease (˜1%) of prepeak 1 of P6MT, P6MTMet, P7GT, and P6GT-P. Lower pHsamples showed more dePEGylation than samples at pH 6 and 7.

Example 17 Agitation/UV Studies

Samples were subjected to stresses that may mimic long term storageconditions and the induction of degradation or aggregation products wasobserved. Degradation upon exposure to UV light may occur in themolecule, or specifically at the oxime or PEG. Two agitation studieswere performed. In the 4 hour agitation study, reconstituted samplesfrom Table 13 were vortexed vigorously at ambient room temperature for 4hours prior to measuring concentration and pH, and analysis wasperformed by SEC-HPLC, RP-HPLC, and SDS-PAGE. A subset of these sampleswere analyzed one week later after storage at 4° C. by SEC-HPLC toinvestigate if aggregates might dissociate back to monomer. Controlsamples were reconstituted samples that were stored at ambient roomtemperature for 4 hours.

In the 2 hour agitation study, polysorbate-free samples, theirpolysorbate-containing counterparts, and P6MA were vortexed gently for 2hours at ambient temperature to confirm the effect of Polysorbate onstability of PEGylated hGH after agitation. See Table 85.

For the UV Exposure study, controls were reconstituted samples stored atambient room temperature for 4 hours. Lyophilized samples were exposedto UV light for 4 hours at ambient temperature and were reconstituted inwater prior to analysis by SDS-PAGE, RP-HPLC, and SEC-HPLC.

See Tables 75-85. SDS-PAGE analysis of these samples is shown in FIGS.61-66. SEC-HPLC data showed that all formulations containing Polysorbate20 at 0.01% induced aggregation during agitation (30-80%). Bam, N B etal. describe the use of Tween 20 (Polysorbate 20) in the inhibition ofinsoluble aggregates during agitation of recombinant hGH (rhGH) (J PharmSci. 1998 December; 87(12):1554-9). Moreover, Maa, Y F et al. indicatedthat polysorbate 20 resulted in a reduction of insoluble proteinaggregates in the production of a spray-dried rhGH powder (J Pharm Sci1998 February; 87(2):152-9). Agitation-induced aggregates were found tobe irreversible non-covalent aggregates. Major light-induced degradationpathways were depegylation and formation of covalent prepeak 2.

TABLE 75 CONCENTRATION, pH AGITATION/UV CONTROLS Formulation Conc. ID(mg/mL) pH P6MT 1.8 6.15 S4MT 1.7 4.03 S5MT 1.6 5.04 S5GT 1.8 5.09 H6MT1.9 5.97 P6GT 2.0 6.04 P6MA 1.9 6.41 P6MS 1.8 6.23 P6MTMet 1.9 6.15 P7MT1.6 6.90 P7GT 1.8 6.88 P6MGT 1.6 6.13 P6MGT-P 1.7 6.11 P6MT-P 1.6 6.13P6GT-P 1.8 6.14

TABLE 76 CONCENTRATION, pH 4 HOUR AGITATION Formulation Conc. ID (mg/mL)pH P6MT 2.0 6.16 S4MT 1.7 3.97 S5MT 1.8 5.02 S5GT 1.9 5.11 H6MT 2.0 5.96P6GT 2.1 6.13 P6MA 2.0 6.39 P6MS 1.9 6.20 P6MTMet 2.0 6.18 P7MT 1.6 6.89P7GT 1.9 6.85 P6MGT 1.8 6.17 P6MGT-P 1.7 6.11 P6MT-P 1.8 6.10 P6GT-P 2.06.18

TABLE 77 CONCENTRATION, pH UV EXPOSURE Formulation Conc. ID (mg/mL) pHP6MT 1.6 6.18 S4MT 1.5 4.06 S5MT 1.6 5.04 S5GT 1.6 5.09 H6MT 1.7 6.04P6GT 1.8 6.10 P6MA 1.8 6.44 P6MS 1.7 6.20 P6MTMet 1.7 6.17 P7MT 1.5 6.96P7GT 1.7 6.93 P6MGT 1.5 6.17 P6MGT-P 1.6 6.14 P6MT-P 1.6 6.15 P6GT-P 1.76.14

TABLE 78 SEC-HPLC (SUMMARY OF PEAK AREAS); AGITATION/UV CONTROLSFormulation % Pre- % Pre- % Main % Un- Total ID peak 1 peak 2 Peak PEGArea P6MT 1.37 1.32 97.14 0.17 4.65E+04 S4MT 1.32 1.93 95.80 0.955.15E+04 S5MT 1.91 1.54 96.17 0.38 4.37E+04 S5GT 1.49 1.72 96.40 0.394.84E+04 H6MT 1.68 1.30 96.84 0.19 4.94E+04 P6GT 1.51 1.57 96.70 0.225.01E+04 P6MA 1.45 1.56 96.85 0.14 5.01E+04 P6MS 1.65 1.35 96.87 0.124.88E+04 P6MTMet 1.22 1.18 97.46 0.14 5.03E+04 P7MT 1.21 1.25 97.39 0.154.30E+04 P7GT 0.89 1.29 97.67 0.16 4.93E+04 P6MGT 1.46 1.35 97.04 0.154.35E+04 P6MGT-P 1.66 1.17 97.01 0.15 4.56E+04 P6MT-P 2.26 1.32 96.240.18 4.45E+04 P6GT-P 1.55 1.46 96.85 0.15 4.79E+04

TABLE 79 SEC-HPLC (SUMMARY OF PEAK AREAS); 4 HOUR AGITATION Formulation% Pre- % Main % Un- Total ID peak Peak PEG Area P6MT 83.90 15.99 0.105.14E+04 S4MT 63.92 35.73 0.35 4.49E+04 S5MT 76.56 23.33 0.11 4.74E+04S5GT 86.15 13.72 0.13 4.93E+04 H6MT 84.89 15.04 0.08 5.35E+04 P6GT 73.7926.08 0.13 5.45E+04 P6MA 28.65 71.22 0.13 5.31E+04 P6MS 47.49 52.37 0.145.13E+04 P6MTMet 82.26 17.64 0.10 4.09E+04 P7MT 53.28 46.55 0.174.27E+04 P7GT 50.31 49.50 0.19 5.05E+04 P6MGT 83.57 16.27 0.16 4.73E+04P6MGT-P 3.49 96.41 0.10 4.51E+04 P6MT-P 5.46 94.38 0.16 4.64E+04 P6GT-P62.52 37.33 0.15 4.98E+04

TABLE 80 SEC-HPLC (SUMMARY OF PEAK AREAS); 4 HOUR AGITATION (REPEAT)Formulation % Pre- % Main % Un- Total ID peak Peak PEG Area P6MT 84.4015.54 0.06 4.21E+04 P6GT 73.20 26.74 0.06 4.53E+04 P6MA 26.51 73.38 0.104.48E+04 P6MGT 83.97 15.89 0.14 3.76E+04 P6MGT-P 2.57 97.31 0.123.75E+04 P6MT-P 4.24 95.62 0.14 3.76E+04 P6GT-P 61.25 38.66 0.083.97E+04

TABLE 81 SEC-HPLC (SUMMARY OF PEAK AREAS); UV EXPOSURE Formulation %Pre- % Pre- % Main % Un- Total ID peak 1 peak 2 Peak PEG Area P6MT 1.561.86 95.57 1.02 4.45E+04 S4MT 1.20 2.59 94.27 1.94 4.20E+04 S5MT 1.691.95 95.43 0.93 4.42E+04 S5GT 1.37 2.11 95.59 0.93 4.62E+04 H6MT 1.601.88 95.65 0.87 4.64E+04 P6GT 1.41 1.99 95.83 0.76 5.03E+04 P6MA 1.302.51 95.26 0.94 5.04E+04 P6MS 1.68 2.01 95.48 0.83 4.68E+04 P6MTMet 1.231.81 96.04 0.92 4.79E+04 P7MT 1.21 2.00 95.61 1.18 4.05E+04 P7GT 1.152.32 94.83 1.70 4.69E+04 P6MGT 1.45 2.00 94.86 1.69 4.34E+04 P6MGT-P1.97 2.52 94.01 1.50 4.28E+04 P6MT-P 2.13 2.50 94.13 1.24 4.58E+04P6GT-P 1.44 2.42 94.70 1.43 4.67E+04

TABLE 82 RP-HPLC (SUMMARY OF PEAK AREAS); AGITATION/UV CONTROLSFormulation % Pre- % Pre- % Post- % Main % Un- Total ID peak 1 peak 2peak Peak PEG Area P6MT 0.36 1.22 1.25 97.09 0.08 6.58E+03 S4MT 0.461.30 2.55 95.28 0.41 6.17E+03 S5MT 0.40 1.41 1.58 96.42 0.19 6.26E+03S5GT 0.35 1.50 1.80 96.32 0.03 7.11E+03 H6MT 0.36 1.36 1.18 97.09 0.026.75E+03 P6GT 0.45 1.51 1.35 96.69 −0.01 7.59E+03 P6MA 0.51 1.41 1.2296.89 −0.04 8.61E+03 P6MS 0.41 1.45 1.18 97.00 −0.04 7.52E+03 P6MTMet0.41 1.41 1.31 96.90 −0.03 7.78E+03 P7MT 0.50 1.42 1.09 97.11 −0.126.33E+03 P7GT 0.46 1.48 1.31 96.74 0.01 7.40E+03 P6MGT 0.45 1.54 1.2396.84 −0.06 6.74E+03 P6MGT-P 0.44 1.47 1.38 96.84 −0.13 6.69E+03 P6MT-P0.41 1.40 1.26 97.07 −0.14 7.32E+03 P6GT-P 0.47 1.52 1.03 97.06 −0.087.25E+03

TABLE 83 RP-HPLC (SUMMARY OF PEAK AREAS); 4 HOUR AGITATION Formulation %Pre- % Pre- % Post- % Main % Un- Total ID peak 1 peak 2 peak Peak PEGArea P6MT 0.41 1.48 1.45 96.70 −0.03 7.55E+03 S4MT 0.58 1.50 2.86 94.700.36 6.42E+03 S5MT 0.46 1.47 1.87 96.21 −0.01 6.97E+03 S5GT 0.56 1.631.98 95.73 0.09 7.34E+03 H6MT 0.43 1.44 1.29 96.88 −0.04 7.93E+03 P6GT0.54 1.63 1.51 96.26 0.05 7.81E+03 P6MA 0.49 1.39 1.19 96.96 −0.038.22E+03 P6MS 0.36 1.42 0.92 97.37 −0.07 7.67E+03 P6MTMet 0.51 1.41 1.4496.65 −0.01 7.91E+03 P7MT 0.63 1.32 1.32 96.85 −0.11 6.69E+03 P7GT 0.491.51 1.48 96.48 0.03 7.81E+03 P6MGT 0.49 1.54 1.19 96.79 −0.01 7.06E+03P6MGT-P 0.38 1.41 1.15 97.12 −0.06 7.53E+03 P6MT-P 0.37 1.39 1.31 96.900.03 7.25E+03 P6GT-P 0.32 1.23 1.08 97.39 −0.02 7.31E+03

TABLE 84 RP-HPLC (SUMMARY OF PEAK AREAS); UV EXPOSURE Formulation % Pre-% Pre- % Post- % Main % Un- Total ID peak 1 peak 2 peak Peak PEG AreaP6MT 0.39 1.33 3.00 93.94 1.34 7.07E+03 S4MT 0.70 1.30 3.60 93.42 0.986.34E+03 S5MT 0.34 1.19 2.49 95.63 0.35 6.93E+03 S5GT 0.30 1.35 2.2895.46 0.61 7.14E+03 H6MT 0.37 1.14 2.22 95.08 1.19 7.92E+03 P6GT 0.301.28 2.36 94.89 1.17 8.43E+03 P6MA 0.32 1.30 1.56 95.68 1.14 7.54E+03P6MS 0.32 1.19 1.40 96.14 0.96 7.78E+03 P6MTMet 0.29 1.14 2.51 94.921.13 7.62E+03 P7MT 0.30 1.31 2.71 94.08 1.60 6.61E+03 P7GT 0.52 1.444.40 91.07 2.57 7.41E+03 P6MGT 0.39 1.47 4.41 91.14 2.59 6.87E+03P6MGT-P 0.31 1.49 3.22 93.04 1.95 6.88E+03 P6MT-P 0.25 1.36 2.77 94.021.60 6.98E+03 P6GT-P 0.36 1.56 3.73 92.20 2.15 6.94E+03

TABLE 85 SEC-HPLC (SUMMARY OF PEAK AREAS); 2 HOUR AGITATION Formulation% Pre- % Main % Un- Total ID Peak Peak PEG Area P6MT 68.19 31.73 0.085.21E+04 P6GT 62.20 37.72 0.08 5.67E+04 P6MA 59.33 40.58 0.09 5.38E+04P6MGT 70.27 29.65 0.07 4.86E+04 P6MGT-P 4.55 95.38 0.07 4.79E+04 P6MT-P17.82 82.10 0.09 4.77E+04 P6GT-P 9.19 90.68 0.13 4.98E+04

Example 18 Agitation Study

Another agitation study was performed with the formulations matrix shownin Table 86. Agitation was performed on 2 mg/mL samples at 250 μL fillin glass vials. Samples were agitated for 4 hours at room temperature.Duplicate set of samples, incubated at room temperature undisturbed, wasused as positive controls (“Controls”). The pH and concentration of thesamples was measured, and SEC-HPLC analysis was performed on t=0,control, and agitated samples as shown in Tables 87-90.

TABLE 86 FORMULATIONS MATRIX Formulation ID Buffer pH Bulking AgentStabilizer Surfactant P7MGT-P 10 mM Phosphate 7 2% Mannitol, 1.25% 2%Trehalose Glycine P7MGT01P 10 mM Phosphate 7 2% Mannitol, 1.25% 2%Trehalose  0.01% PS20 Glycine P7MGT005P 10 mM Phosphate 7 2% Mannitol,1.25% 2% Trehalose 0.005% PS20 Glycine P7MGT0025P 10 mM Phosphate 7 2%Mannitol, 1.25% 2% Trehalose 0.0025% PS20  Glycine P7MGT001P 10 mMPhosphate 7 2% Mannitol, 1.25% 2% Trehalose 0.001% PS20 Glycine H7MT-P10 mM Histidine 7 4% Mannitol 2% Trehalose H7MGT-P 10 mM Histidine 7 2%Mannitol, 1.25% 2% Trehalose Glycine 7MHT-P 7 2% Mannitol, 1.25% 2%Trehalose Histidine

TABLE 87 CONCENTRATION, pH Formulation Conc. ID (mg/mL) pH P7MGT-P 2.07.14 P7MGT01P 1.9 7.20 P7MGT005P 1.8 7.20 P7MGT0025P 2.0 7.19 P7MGT001P2.0 7.22 H7MT-P 2.0 6.83 H7MGT-P 2.1 6.82 7MHT-P 2.3 6.91

TABLE 88 SEC-HPLC (SUMMARY OF PEAK AREAS); AGITATION T = 0 Formulation %Pre- % Pre- % Main % Un- Total ID peak 1 peak 2 Peak PEG Area P7MGT-P0.57 0.67 98.68 0.08 5.72E+04 P7MGT01P 0.45 0.58 98.91 0.06 5.75E+04P7MGT005P 0.63 0.66 98.65 0.07 5.89E+04 P7MGT0025P 0.46 0.59 98.84 0.115.94E+04 P7MGT001P 0.49 0.59 98.84 0.07 6.13E+04 H7MT-P 0.66 0.72 98.560.06 5.78E+04 H7MGT-P 0.67 0.68 98.57 0.08 6.27E+04 7MHT-P n/a n/a n/an/a n/a

TABLE 89 SEC-HPLC (SUMMARY OF PEAK AREAS): AGITATION CONTROLSFormulation % Pre- % Pre- % Main % Un- Total ID peak 1 peak 2 Peak PEGArea P7MGT-P 0.57 0.66 98.71 0.06 6.28E+04 P7MGT01P 0.46 0.61 98.86 0.085.96E+04 P7MGT005P 0.46 0.61 98.83 0.10 5.51E+04 P7MGT0025P 0.42 0.5998.90 0.08 6.06E+04 P7MGT001P 0.48 0.57 98.88 0.08 5.95E+04 H7MT-P 0.600.62 98.71 0.07 5.89E+04 H7MGT-P 0.67 0.61 98.61 0.11 6.08E+04 7MHT-P0.63 0.72 98.57 0.07 6.48E+04

TABLE 90 SEC-HPLC (SUMMARY OF PEAK AREAS); AFTER 4 HOUR AGITATIONFormulation % Pre- % Pre- % Main % Un- Total ID peak 1 peak 2 Peak PEGArea P7MGT-P 0.57 0.65 98.67 0.11 5.80E+04 P7MGT01P 0.00 19.03 80.870.10 6.07E+04 P7MGT005P 0.00 24.37 75.54 0.09 5.90E+04 P7MGT0025P 0.0013.55 86.37 0.08 6.07E+04 P7MGT001P 0.00 7.54 92.36 0.10 6.13E+04 H7MT-P0.80 0.69 98.39 0.11 5.52E+04 H7MGT-P 0.71 0.68 98.51 0.10 6.04E+047MHT-P 0.59 0.70 98.61 0.09 6.55E+04

Trace amounts of polysorbate induced aggregation under agitation. Whenagitated, Histidine formulations performed comparably toglycine-containing formulations.

Example 19 Accelerated Aggregation Studies

Accelerated stress studies were performed to mimic long term storageconditions and identify potential degradation or aggregation products.Accelerated aggregation studies were performed using 2 formulations(Formulation ID H7MT-P and H7MGT-P) at approximately 8 and 14 mg/ml. Thecomponents of the formulations are described in Table 91. The “-P”designation indicates no polysorbate 20. Stresses studied werefreeze-thaw conditions, agitation, UV exposure, and temperature. Themethods described above were used to evaluate PEGylated hGH. Sampleswere evaluated by SDS-PAGE and SEC-HPLC methods described in Examples 9and 11, respectively. Additional time points for analysis include 1month, 2 month, etc. Additional techniques for analysis of samplesinclude Dynamic Light Scattering (DLS) and AnalyticalUltracentrifugation.

TABLE 91 FORMULATIONS MATRIX (ACCELERATED AGGREGATION STUDIES)Formulation ID Buffer pH Bulking Agent Stabilizer Concentration H7MT-P10 mM Histidine 7 4% Mannitol 2% Trehalose  8 mg/mL H7MT-P 10 mMHistidine 7 4% Mannitol 2% Trehalose 14 mg/mL H7MGT-P 10 mM Histidine 72% Mannitol, 1.25% 2% Trehalose  8 mg/mL Glycine H7MGT-P 10 mM Histidine7 2% Mannitol, 1.25% 2% Trehalose 14 mg/mL Glycine

63 mL of 5.7 mg/mL PEGylated hGH (in 20 mM sodium citrate, 2% glycine,0.5% mannitol, pH 6) was concentrated to 14.2 mg/mL. Half of theconcentrate was dialyzed against H7MT-P, and the other half againstH7MGT-P. Since the dialysis caused the protein to be diluted, the samplein H7MT-P was concentrated to 13.6 mg/mL and the H7MGT-P wasconcentrated to 13.5 mg/mL. A portion of the PEGylated hGH(approximately, 4.5 mL) in H7MT-P at 13.6 mg/mL was diluted to 7.8 mg/mLwith H7MT-P buffer. Similarly approximately 4.5 mL of PEGylated hGH inH7MGT-P at 13.5 mg/mL was diluted to 8.0 mg/mL with H7MGT-P buffer.

Freeze/Thaw Studies

The formulation was frozen at −70° C. for 15 minutes. It was then thawedat 25° C. The vial was then uncapped to remove a portion of the samplefor SEC-HPLC and SDS-PAGE analysis, and the vial re-capped. Thisprocedure was repeated five times. The results of the SEC-HPLC analysisare shown in Tables 92-95. SDS-PAGE analysis is shown in FIGS. 67-70.

TABLE 92 SEC-HPLC (SUMMARY OF PEAK AREAS); FORMULATION ID: H7MT-P; Conc:8 mg/mL % Pre- % Pre- % Main % Un- Total Frequency F/T peak 1 peak 2Peak PEG Area 0 1.00 0.71 98.15 0.14 7.16E+04 1 0.70 0.65 98.57 0.087.50E+04 2 0.75 0.65 98.53 0.07 7.57E+04 3 0.78 0.66 98.43 0.12 7.69E+044 0.88 0.68 98.36 0.08 7.62E+04 5 1.04 0.77 98.10 0.08 7.89E+04

TABLE 93 SEC-HPLC (SUMMARY OF PEAK AREAS); FORMULATION ID: H7MT-P; Conc:14 mg/mL % Pre- % Pre- % Main % Un- Total Frequency F/T peak 1 peak 2Peak PEG Area 0 0.66 0.62 98.63 0.09 6.81E+04 1 0.64 0.63 98.65 0.087.36E+04 2 0.65 0.63 98.64 0.08 7.35E+04 3 0.67 0.66 98.59 0.08 7.96E+044 0.76 0.66 98.47 0.10 8.08E+04 5 0.84 0.65 98.39 0.11 7.60E+04

TABLE 94 SEC-HPLC (SUMMARY OF PEAK AREAS); FORMULATION ID: H7MGT-P;Conc: 8 mg/mL % Pre- % Pre- % Main % Un- Total Frequency F/T peak 1 peak2 Peak PEG Area 0 0.73 0.64 98.51 0.12 7.41E+04 1 0.61 0.65 98.62 0.127.45E+04 2 0.77 0.62 98.47 0.14 7.42E+04 3 0.82 0.67 98.41 0.10 7.58E+044 0.69 0.67 98.55 0.08 7.58E+04 5 0.92 0.69 98.26 0.13 7.43E+04

TABLE 95 SEC-HPLC (SUMMARY OF PEAK AREAS); FORMULATION ID: H7MGT-P;Conc: 14 mg/mL % Pre- % Pre- % Main % Un- Total Frequency F/T peak 1peak 2 Peak PEG Area 0 0.81 0.66 98.43 0.09 7.26E+04 1 0.78 0.68 98.440.10 7.64E+04 2 0.75 0.65 98.47 0.13 7.44E+04 3 0.74 0.68 98.49 0.088.06E+04 4 0.89 0.65 98.37 0.09 7.42E+04 5 0.99 0.70 98.21 0.11 7.16E+04

The freeze/thawing of the samples caused a 0.2-0.3% increase in higherMW aggregates, with possible dissociation of higher MW aggregates afterthe first freeze-thaw cycle in 8 mg/mL samples.

Vortex/UV Exposure

For this study, the formulations matrix shown in Table 91 was used. Forthe control samples in the agitation (vortex) and UV exposure studies,formulations were held at room temperature for 6 hours. In the agitationstudy, liquid samples were vortexed at room temperature for 6 hours athigh speed. In the UV exposure study, lyophilized samples were exposedto UV light at room temperature for 4 hours and reconstituted prior toanalysis. Samples were removed for SEC-HPLC and SDS-PAGE analysis.Tables 96-98 show the SEC-HPLC data for the control samples, thevortexed samples, and the UV-exposed samples. SDS-PAGE analysis of thesesamples is shown as FIGS. 71 and 72.

TABLE 96 SEC-HPLC (SUMMARY OF PEAK AREAS); VORTEX/UV CONTROLSFormulation % Pre- % Pre- % Main % Un- Total ID peak 1 peak 2 Peak PEGArea H7MT-P; 8 mg/mL 0.78 0.68 98.43 0.11 7.58E+04 H7MT-P; 14 mg/mL 0.660.66 98.57 0.11 7.63E+04 H7MGT-P; 8 mg/mL 0.69 0.69 98.49 0.13 7.52E+04H7MGT-P; 14 mg/mL 0.77 0.68 98.43 0.12 7.58E+04

TABLE 97 SEC-HPLC (SUMMARY OF PEAK AREAS); VORTEXED SAMPLES (vortexedfor 6 hours at room temperature) Formulation % Pre- % Pre- % Main % Un-Total ID Peak 1 peak 2 Peak PEG Area H7MT-P; 8 mg/mL 9.99 n/a 89.89 0.117.68E+04 H7MT-P; 14 mg/mL 14.75 n/a 85.13 0.12 7.11E+04 H7MGT-P; 8 mg/mL16.85 n/a 83.04 0.10 7.78E+04 H7MGT-P; 14 mg/mL 4.22 n/a 95.69 0.097.40E+04

TABLE 98 SEC-HPLC (SUMMARY OF PEAK AREAS); UV- EXPOSED SAMPLES (exposedto UV light for 4 hours at room temperature) Formulation % Pre- % Pre- %Main % Un- Total ID peak 1 peak 2 Peak PEG Area H7MT-P; 8 mg/mL 0.640.91 97.96 0.49 7.49E+04 H7MT-P; 14 mg/mL 0.67 0.84 98.17 0.32 7.14E+04H7MGT-P; 8 mg/mL 0.53 0.82 98.14 0.50 7.55E+04 H7MGT-P; 14 mg/mL 0.710.83 98.13 0.33 7.38E+04

Histidine formulations without polysorbate 20 were confirmed to reducethe amount of aggregates formed with vigorous agitation.

DePEGylation was the main degradation event and was more pronounced insamples with the lower concentration of hGH. UV exposure resulted in anincrease in % Pre-peak 2 (dimerization) compared to the control.

In another agitation study, lyophilized samples were stored at 4° C. or40° C. for 1 week or 2 weeks. The samples were then reconstituted priorto analysis by SDS-PAGE and SEC-HPLC. Tables 99-102 show the SEC-HPLCresults. FIGS. 73-74 show SDS-PAGE analysis for 1 week samples. FIGS.75-76 show SDS-PAGE analysis for 2 week samples.

TABLE 99 SEC-HPLC (SUMMARY OF PEAK AREAS); T = 1 week, 4° C. Formulation% Pre- % Pre- % Main % Un- Total ID peak 1 peak 2 Peak PEG Area H7MT-P;8 mg/mL 0.63 0.65 98.59 0.13 7.45E+04 H7MT-P; 14 mg/mL 0.51 0.60 98.760.12 7.41E+04 H7MGT-P; 8 mg/mL 0.62 0.63 98.60 0.14 7.46E+04 H7MGT-P; 14mg/mL 0.81 0.63 98.44 0.12 7.42E+04

TABLE 100 SEC-HPLC (SUMMARY OF PEAK AREAS); T = 2 week, 4° C.Formulation % Pre- % Pre- % Main % Un- Total ID peak 1 peak 2 Peak PEGArea H7MT-P; 8 mg/mL 0.72 0.64 98.55 0.09 7.35E+04 H7MT-P; 14 mg/mL 0.580.66 98.69 0.07 7.12E+04 H7MGT-P; 8 mg/mL 0.42 0.51 98.98 0.09 7.18E+04H7MGT-P; 14 mg/mL 0.50 0.66 98.75 0.08 7.15E+04

TABLE 101 SEC-HPLC (SUMMARY OF PEAK AREAS); T = 1 week, 40° C.Formulation % Pre- % Pre- % Main % Un- Total ID peak 1 peak 2 Peak PEGArea H7MT-P; 8 mg/mL 0.51 0.87 96.83 1.79 7.65E+04 H7MT-P; 14 mg/mL 0.450.92 96.83 1.80 7.19E+04 H7MGT-P; 8 mg/mL 0.41 0.77 96.34 2.48 7.82E+04H7MGT-P; 14 mg/mL 0.46 0.88 96.11 2.55 7.40E+04

TABLE 102 SEC-HPLC (SUMMARY OF PEAK AREAS); T = 2 week, 40° C.Formulation % Pre- % Pre- % Main % Un- Total ID peak 1 peak 2 Peak PEGArea H7MT-P; 8 mg/mL 0.39 0.90 95.80 2.91 7.41E+04 H7MT-P; 14 mg/mL 0.391.02 95.37 3.22 6.97E+04 H7MGT-P; 8 mg/mL 0.32 0.77 94.50 4.41 7.36E+04H7MGT-P; 14 mg/mL 0.39 0.97 94.15 4.49 6.89E+04

After 1 week, H7MT-P produced less higher molecular weight aggregatethan H7MGT-P at 4° C. H7MT-P resulted in slightly more dimer formation,but less dePEGylated hGH than H7MGT-P at 40° C. After two weeks, anincreased amount of dimerization was observed at 40° C. Significantde-PEGylation was observed at 40° C. H7MT-P formed less de-PEGylated hGHthan H7MGT-P at 40° C. Additional time points include measurements at 4weeks.

In addition, samples of PEGylated-hGH were exposed to thermal-unfoldingconditions. The thermal-unfolding samples were incubated for 10 minutesat 85° C., which is above the melting temperature (T_(m)) of 82° C.Thermal-unfolding induced aggregation. Table 103 shows the SEC-HPLCanalysis of the samples exposed to thermal-unfolding. SDS-PAGE analysisis shown in FIGS. 73-74.

TABLE 103 SEC-HPLC (SUMMARY OF PEAK AREAS); THERMAL-UNFOLDINGFormulation % Pre- % Pre- % Main % Un- Total ID peak 1 peak 2 Peak PEGArea H7MT-P; 8 mg/mL 89.23 n/a 10.77 0 8.14E+04 H7MT-P; 14 mg/mL 91.89n/a 8.11 0 7.81E+04 H7MGT-P; 8 mg/mL 90.09 n/a 9.91 0 8.42E+04 H7MGT-P;14 mg/mL 92.18 n/a 7.82 0 7.97E+04

Example 20 FT/IR (Fourier Transform Infrared Spectroscopy) Scans

Analysis of PEGylated hGH was performed with FT/IR. FT/IR scans wereperformed on a Jasco model FT/IR 660 Plus. Each sample was scanned 320times at a resolution of 4 cm⁻¹ and analyzed with Jasco Spectra Managerv1.53.00. Air backgrounds and water blanks were taken before each day'ssample runs. The elimination of signals derived from water was confirmedby the absence of absorbance at 1700 cm⁻¹. After the appropriatebackground and blank controls were subtracted from each sample spectrum,second derivative analysis of the remaining protein and excipient peakswas performed in the Amide I region (1600-1700 cm⁻¹). As a liquidcontrol, PEG-hGH bulk at 6 mg/mL was used.

The Amide I signal of PEG-hGH was relatively weaker than other proteinseven after lyophilization. However, most samples showed α-helix signalat 1651 cm⁻¹, so qualitative comparison among formulation candidatescould be accomplished. Glycine shows a strong signal at Amide I region,so the signal could have been distorted during the course of subtractingglycine signal from original data.

Second-derivative FTIR spectra [signal intensity (y axis) vs. wavenumber in cm⁻¹ (x-axis)] of the P6MT, S4MT, S5MT, H6MT, P6GT, P6MS,P6MTmet, P7MT, and P6MT-P formulations compared to the liquid controlshowed: 1) Most mannitol formulations preserved good α-helix signalregardless of buffer or pH. 2) One formulation, P6GT containing glycine,showed significant shift in the band signal. 3) Other peaks wereobserved at 1620, 1630, 1725, and 1750. Second derivative FTIR spectraof the S5GT, P6MA, P7GT, P6MGT, P6MGT-P, and P6GT-P formulations hadpoor α-helix signal. Moreover, all glycine formulations showedsignificant deviation from the native signal of liquid sample. Thearginine-containing formulation exhibited major structural changesduring lyophilization.

After two months of storage of the lyophilized material at 4° C., FT/IRwas performed. The Amide I signal of PEG-hGH was relatively weaker thanother proteins even after lyophilization. However, most samples showedα-helix signal at 1651 cm⁻¹, so qualitative comparison among formulationcandidates could be accomplished. Glycine showed a strong signal atAmide I region. At two months, all glycine formulations showedsignificant deviation from the native signal of liquid sample. Mostmannitol formulations preserved good α-helix signal regardless of bufferor pH.

Example 21 Agitation (Surfactant Testing)

PEG-hGH in 20 mM sodium citrate, 2% glycine, 0.5% mannitol, pH 6 wasdiluted to 2 mg/mL with H7MT-P. 0.01% polysorbate 20 (PS) was added toone sample, 0.01% Pluronic F68 (F68) to another, and the third had nosurfactant added. Agitation was performed with 500 μL fill in glassvials. Samples were agitated for 1 hour at room temperature. SEC-HPLCanalysis of the samples is shown in Table 104; samples that wereagitated are noted with “Vtx.”.

TABLE 104 SEC-HPLC (SUMMARY OF PEAK AREAS); AGITATION WITH SURFACTANT %Pre- Formulation peaks % Main % Un- Total ID 1 + 2 Peak PEG Area H7MT-P,t = 0 2.84 98.16 0 5.81E+04 H7MT, 0.01% PS, t = 0 1.49 98.51 0 5.59E+04H7MT, 0.01% F68, t = 0 1.58 98.42 0 5.75E+04 H7MT-P, 1 hr Vtx 12.5487.46 0 5.79E+04 H7MT, 0.01% PS, 1 hr Vtx 57.21 42.79 0 6.13E+04 H7MT,0.01% F68, 1 hr Vtx 4.43 95.57 0 5.58E+04

Relative to the other samples tested, Pluronic F68 was the mosteffective in preventing agitation induced aggregation as shown in Table104.

Additional experiments may include, dialyzing the samples instead ofdiluting them to remove residual excipients and/or testing othersurfactants, including but not limited to, polysorbate 80. A surfactantmay be used alone or in combination with one or more other surfactants.Additional studies include testing the effect of the followingsurfactants on the stability of PEG-hGH in H7MT formulation againstagitation-induced aggregation: 1) no surfactant (negative control); 2)polysorbate 20 at various amounts including, but not limited to, 0.01%;3) Pluronic F68 (or Poloxamer 188) at various amounts including, but notlimited to, 0.005%, 0.01%, 0.05%, and 0.1%; 4) polysorbate 80 at variousamounts including, but not limited to, 0.01%; and 5) a combination ofpluronic F68 and polysorbate 20 or pluronic F68 and other surfactants.Additional lyophilized formulations (H7MT) containing the bestsurfactant or combination of surfactants will be prepared and theirstability at 40° C. will be compared to the H7MT formulation. Theconcentration of PEG-hGH will be 2 mg/ml, and time points for analysiswill be at 0, 1, 2, 3, and 4 weeks, or longer. The techniques describedherein will be used for analysis.

Example 22 Long Term Studies

Additional long term stability studies include evaluation offormulations after storage at various temperatures, including but notlimited to, 4° C. and 29° C., for 0, 3, 6, 9, 12, 18, and 24 months. Thestability of samples reconstituted after lyophilization may beinvestigated during storage at 2-8° C. for various lengths of time, suchas 0, 1 day, 3 days, and 1 week.

Example 23 Lyophilized Formulation Study

Additional data was generated from the lyophilized formulation studydiscussed in Example 14. Data generated for the four month time pointinclude: SDS-gels (samples stored at 4° C. and 25° C. for 4 months;FIGS. 77-80); concentration and pH post reconstitution (Tables 105-106);SEC-HPLC analysis of samples stored at 4° C. and 25° C. for 4 months(Tables 107-108); and RP-HPLC analysis of samples stored at 4° C. and25° C. for 4 months (Tables 109-110). After four months, the formulationcontaining histidine continued to show the least amount of covalentaggregates via SDS-PAGE of the formulations tested. Over the last twomonths, H6MT and P7MT have showed minimal change at 25° C.

TABLE 105 CONCENTRATION, pH POST RECONSTITUTION; T = 4 months; 4° C.Formulation Conc. ID (mg/mL) pH P6MT 1.8 6.25 H6MT 2.5 6.09 P6GT 2.06.20 P6MS 1.8 6.29 P6MTMet 1.8 6.24 P7MT 1.6 7.04 P7GT 1.8 7.08 P6MGT1.7 6.25 P6MGT-P 1.7 6.25 P6MT-P 1.7 6.20 P6GT-P 1.3 6.30

TABLE 106 CONCENTRATION, pH POST RECONSTITUTION; T = 4 months; 25° C.Formulation Conc. ID (mg/mL) pH P6MT 1.5 6.28 H6MT 1.9 6.06 P6GT 1.96.24 P6MS 1.9 6.28 P6MTMet 1.8 6.18 P7MT 1.6 7.10 P7GT 1.8 6.99 P6MGT1.7 6.23 P6MGT-P 1.7 6.18 P6MT-P 1.6 6.23 P6GT-P 1.7 6.21

TABLE 107 SEC-HPLC (SUMMARY OF PEAK AREAS); T = 4 month; 4° C.Formulation % Pre- % Pre- % Main % Un- Total ID peak 1 peak 2 Peak PEGArea P6MT 0.54 1.17 98.29 0.00 5.03E+04 H6MT 0.58 1.11 98.30 0.006.96E+04 P6GT 0.48 1.40 98.12 0.00 5.70E+04 P6MS 0.57 1.08 98.35 0.005.20E+04 P6MTMet 0.50 1.11 98.39 0.00 5.16E+04 P7MT 0.46 1.13 98.41 0.004.59E+04 P7GT 0.42 1.42 98.16 0.00 5.11E+04 P6MGT 0.49 1.07 98.44 0.004.67E+04 P6MGT-P 0.52 1.04 98.44 0.00 4.72E+04 P6MT-P 0.76 1.30 97.940.00 4.71E+04 P6GT-P 0.75 1.57 97.67 0.00 3.59E+04

TABLE 108 SEC-HPLC (SUMMARY OF PEAK AREAS); T = 4 month; 25° C. % Pre- %Pre- % Main % Un- Total Formulation peak 1 peak 2 Peak PEG Area P6MT0.59 1.88 97.53 0.00 4.20E+04 H6MT 0.76 1.44 97.80 0.00 5.27E+04 P6GT0.49 2.34 97.17 0.00 5.33E+04 P6MS 0.71 2.80 96.49 0.00 5.57E+04 P6MTMet0.58 2.97 96.45 0.00 5.10E+04 P7MT 0.39 1.63 97.98 0.00 4.54E+04 P7GT0.36 2.10 97.54 0.00 5.12E+04 P6MGT 0.50 2.68 96.82 0.00 4.62E+04P6MGT-P 0.57 2.58 96.85 0.00 4.74E+04 P6MT-P 0.96 2.13 96.92 0.004.58E+04 P6GT-P 0.71 2.34 96.95 0.00 4.98E+04

TABLE 109 RP-HPLC (SUMMARY OF PEAK AREAS); T = 4 month; 4° C.Formulation % Pre- % Pre- % Post- % Main % Un- Total ID peak 1 peak 2peak Peak PEG Area P6MT 0.17 0.90 1.12 97.63 0.18 8.36E+03 H6MT 0.160.78 0.88 98.06 0.12 1.16E+04 P6GT 0.26 0.87 1.39 97.20 0.28 9.24E+03P6MS 0.16 0.83 0.84 98.10 0.06 8.61E+03 P6MTMet 0.12 0.87 1.14 97.710.14 8.52E+03 P7MT 0.17 0.79 1.24 97.75 0.06 7.60E+03 P7GT 0.26 1.031.88 96.44 0.39 8.26E+03 P6MGT 0.17 1.03 1.40 97.01 0.38 7.78E+03P6MGT-P 0.16 0.96 1.23 97.42 0.23 7.60E+03 P6MT-P 0.20 0.91 1.26 97.430.20 7.62E+03 P6GT-P 0.19 1.21 1.61 96.86 0.13 6.97E+03

TABLE 110 RP-HPLC (SUMMARY OF PEAK AREAS); T = 4 month; 25° C.Formulation % Pre- % Pre- % Post- % Main % Un- Total ID peak 1 peak 2peak Peak PEG Area P6MT 0.25 1.10 2.46 95.74 0.44 6.74E+03 H6MT 0.590.99 3.05 94.51 0.86 8.56E+03 P6GT 0.25 1.53 2.36 95.38 0.48 8.55E+03P6MS 0.28 1.06 1.84 96.60 0.22 8.82E+03 P6MTMet 0.27 1.39 5.63 90.482.22 8.21E+03 P7MT 0.20 0.90 1.47 97.22 0.20 7.17E+03 P7GT 0.22 1.371.72 96.33 0.36 8.01E+03 P6MGT 0.35 1.84 2.75 94.40 0.66 7.64E+03P6MGT-P 0.28 2.05 2.76 94.46 0.44 7.78E+03 P6MT-P 0.32 1.06 2.33 95.810.48 7.47E+03 P6GT-P 0.29 1.46 2.58 95.16 0.51 7.81E+03

Example 24 Histidine Interaction Study with PEG-HGH

The objective of this study is to investigate the pH drop that has beenobserved in 10 mM histidine buffer, 4% mannitol, 2% trehalose, pH 7.0when PEG-hGH is added at concentrations of at least 8 mg/mL. This studyinvestigated whether the concentration dependent pH change in histidineformulations is due to the binding of histidine with PEG-hGH.

RP-HPLC and/or IEX-HPLC methods for determining free/bound histidine maybe used in such studies.

Dialysis of 5 mg/mL and 25 mg/mL PEG-hGH

One or two concentrations of PEG-hGH were dialyzed against one of thefollowing buffers: 10 mM Na₂HPO₄, pH 7.1; 10 mM histidine (pH 7.0); 10mM histidine (free base), pH 7.7; 30 mM histidine (free base), pH 7.7.The pH of the protein was measured after dialysis. The amount ofhistidine was determined by running SEC-HPLC and measuring the histidinepeak which elutes after protein peak. The 214 nm:280 nm was compared todetermine if histidine bound to the PEG-hGH.

For 10 mM Histidine (free base), pH 7.7, the 4.5 mg/ml protein samplehad a pH of 7.18, and the 14.0 mg/ml protein sample had a pH of 6.89.The 214 nm:280 nm ratios were the same (19.2) for both proteinconcentrations. For 30 mM Histidine (free base), pH 7.7, the 13.3 mg/mlprotein sample had a pH of 7.19. The 214 nm:280 nm ratio was 1.9.2. For10 mM His, pH 7.0, the 4.6 mg/ml protein sample had a pH of 6.94, andthe 13.2 mg/ml protein sample had a pH of 6.76. The 214 nm:280 nm ratioswere the same (19.1) for both protein concentrations. For 10 mM Na₂HPO₄,pH 7.1, the 4.6 mg/ml protein sample had a pH of 7.10, and the 13.2mg/ml protein sample had a pH of 7.09. The 214 nm:280 nm ratios were19.2 for the lower protein concentration, and 19.1 for the higherprotein concentration.

pH Change During Concentration

pH changes were measured of the protein during concentration. 10 mMhistidine (free base) and 30 mM His (free base) were tested; the proteinconcentration started at 1 mg/ml. With 10 mM His (free base), pH 7.7,the following results were found: 1.0 mg/ml protein had pH 7.41; 11.7mg/ml protein had pH 6.82; and 15.0 mg/ml protein had pH 6.71. With 30mM His (free base), pH 7.7, the following results were found: 1.0 mg/mlprotein had pH 7.58; 11.4 mg/ml protein had pH 7.22; and 17.1 mg/mlprotein had pH 7.05.

Addition of Histidine

PEG-hGH was concentrated with 14 mg/ml and dialyzed against water. Thehistidine concentration was increased by adding small volumes ofconcentrated histidine to the protein. The pH was measured at eachhistidine increase. The following pH measurements were determined withthe histidine concentration (mM): pH 5.57 at 0 mM Histidine, pH 5.68 at0.25 mM Histidine, pH 5.74 at 0.5 mM Histidine, pH 5.86 at 1 mMHistidine, pH 6.13 at 2.5 mM Histidine, pH 6.36 at 5 mM Histidine, pH6.63 at 10 mM Histidine, and pH 7.05 at 30 mM Histidine.

In these studies, it was found that histidine does not bind to thePEG-hGH. The buffering capacity of histidine was unable to overcome thebuffering capacity of the protein. Monobasic phosphate is tested todetermine if it can provide enough buffering capacity to maintain pH.Any buffer that has a buffering capacity between about pH 5.5 and aboutpH 8.0, including but not limited to, monobasic phosphate may besuitable. Formulations with histidine had lower amounts of covalentaggregates and aggregates from agitation.

Monobasic phosphate is tested in the following assay. PEG-hGH isconcentrated to 14 mg/ml and dialyzed against water. The phosphateconcentration is increased by adding small volumes of concentratedphosphate to the protein. The pH is measured at each phosphate increase,and a plot is generated with pH vs. phosphate concentration (mM)

Example 25

Additional data was generated from the accelerated aggregation studydiscussed in Example 19. Data generated for the four week time pointinclude: SDS-gels (samples stored at 4° C. and 40° C. for 4 weeks; FIGS.81-82); pH (Table 111); and SEC-HPLC analysis of samples stored at 4° C.and 40° C. for 4 weeks (Tables 112-113).

TABLE 111 Measured pH Formulation ID Concentration 4° C. pH 40° C. pHH7MT-P  8 mg/mL 6.32 6.29 H7MT-P 14 mg/mL 6.17 6.17 H7MGT-P  8 mg/mL6.42 6.39 H7MGT-P 14 mg/mL 6.28 6.25

TABLE 112 SEC-HPLC (SUMMARY OF PEAK AREAS); T = 4 week, 4° C.Formulation % Pre- % Pre- % Main % Un- Total ID peak 1 peak 2 Peak PEGArea H7MT-P 8 mg/mL 0.57 0.72 98.56 0.15 8.15E+04 H7MT-P 14 mg/mL 0.470.69 98.68 0.16 7.92E+04 H7MGT-P 8 mg/mL 0.50 0.69 98.60 0.21 7.83E+04H7MGT-P 14 mg/mL 0.64 0.71 98.44 0.21 7.78E+04

TABLE 113 SEC-HPLC (SUMMARY OF PEAK AREAS); T = 4 week, 40° C.Formulation % Pre- % Pre- % Main % Un- Total ID peak 1 peak 2 Peak PEGArea H7MT-P 8 mg/mL 0.35 1.08 92.00 6.58 8.56E+04 H7MT-P 14 mg/mL 0.291.24 91.53 6.94 8.07E+04 H7MGT-P 8 mg/mL 0.35 0.92 89.66 9.06 8.05E+04H7MGT-P 14 mg/mL 0.37 1.15 89.31 9.17 7.61E+04

After the four week time period, H7MT-P formed less de-PEGylated hGHthan H7MGT-P at both 4° C. and 40° C., and a small amount of covalentaggregates at 40° C. Overall, at 40° C., the main degradation productwas de-PEGylated hGH, and there was an increased amount of dimerizationand more covalent aggregates than at 4° C.

Example 26 Agitation Study (Surfactants)

A follow-up study was performed to those described in Example 21.PEG-hGH was dialyzed with H7MT, and the dialyzed protein was diluted to2 mg/ml with H7MT. Various amounts of the surfactant Pluronic F68 wereadded to the samples: no Pluronic F68, 0.01% Pluronic F68, 0.05%Pluronic F68, 0.1% Pluronic F68, 0.25% Pluronic F68, or 0.5% PluronicF68. Two control samples contained H7MT buffer with 0.01% Pluronic Acidwithout protein. One sample was vortexed, whereas the other one was not(t=0). Agitation was performed with 500 uL fill in glass vials, andsamples were agitated for 2 hours at room temperature. SEC-HPLC analysiswas performed on the various samples, and the results are shown in Table114.

TABLE 114 SEC-HPLC (SUMMARY OF PEAK AREAS); Agitation % Pre- % MainTotal Formulation ID peak 1 + 2 Peak Area H7MT, no surfactant, t = 04.40 95.60 8.85E+04 H7MT, no surfactant, 2 hr. vortex 12.45 87.557.47E+04 H7MT, 0.01% F68, 2 hr. vortex 15.66 84.34 7.38E+04 H7MT, 0.05%F68, 2 hr. vortex 2.45 97.55 7.11E+04 H7MT, 0.1% F68, 2 hr. vortex 1.6998.31 7.12E+04 H7MT, 0.25% F68, 2 hr. vortex 1.37 98.63 7.35E+04 H7MT,0.5% F68, 2 hr. vortex 1.36 98.64 7.33E+04

In a study investigating aggregate reversibility, 0.1% Pluronic F68 wasadded to the vortexed control (no surfactant) sample to examine whetheragitation-induced aggregates could be dissociated by Pluronic F68. Thesamples were analyzed by SEC-HPLC. The results of this study showed thatPluronic F68 can reduce agitation-induced aggregation, but does notdissociate it. H7MT with 0.1% Pluronic F68 had a smaller amount ofaggregate than the formulations with lower amounts of Pluronic F68.

Example 27 Sedimentation Velocity Analysis

This analysis was performed to measure the aggregation of the followingthree samples: PEG-hGH polypeptide at 39.9 mg/ml, 24.3 mg/ml, and 1.0mg/ml. These stocks were diluted to 0.6 mg/ml immediately prior tomeasurement with Dulbecco's phosphate buffered saline (Gibco part no.144190-144). The high-resolution sedimentation coefficient distributionsfor these samples were generated with the vertical axis giving theconcentration and the horizontal axis showing the separation on thebasis of sedimentation coefficient. Each distribution was normalized toaccount for concentration differences among the samples. PEG-hGH at 39.9mg/ml had its main peak at 1.27 S, and that peak represented 98.7% ofthe total absorbance. PEG-hGH at 24.3 mg/ml had its main peak at 1.29 S,and that peak represented 97.5% of the total absorbance. Minor peaksthat sediment faster than the main peak (monomer) were found.

Example 28 AUC/SEC Study

Samples with different concentrations of PEGylated hGH polypeptide (39.9mg/ml, 24.3 mg/ml, and 1.1 mg/ml) and the formulations shown in Table115 were found to show similar amounts of aggregation by SEC-HPLC,SDS-PAGE, and AUC. Table 116 shows SEC-HPLC results from 20 ug ofmaterial loaded onto the column. SDS-PAGE analysis (10 ug loaded in eachlane) is shown as FIG. 83.

TABLE 115 Buffer pH Bulking Agent Stabilizer Concentration 30 mMHistidine 7 4% Mannitol 2% Trehalose 39.9 mg/mL 30 mM Histidine 7 4%Mannitol 2% Trehalose 24.3 mg/mL 10 mM Histidine 7 4% Mannitol 2%Trehalose  1.1 mg/mL

TABLE 116 % Pre- % Pre- % Main % Un- Total Concentration peak 1 peak 2Peak PEG Area 39.9 mg/mL 0.56 1.46 97.98 0.00 5.98E+04 24.3 mg/mL 0.481.46 98.06 0.00 7.55E+04  1.1 mg/mL 0.67 1.55 97.79 0.00 7.58E+04

Example 29 Intermediate Stability Study

Table 117 details the intermediate stability study. Formulation IDH7.3MT is 30 mM Histidine, 4% Mannitol, 2% Trehalose, pH 7.3.Formulation ID H7.3MT+F is 30 mM Histidine, 4% Mannitol, 2% Trehalose,pH 7.3 with 0.1% Pluronic F68. Formulation ID HP7MT is 10 mM Histidine,10 mM Phosphate, 4% Mannitol, 2% Trehalose, pH 7.0. Formulation IDHP7MT+F is 10 mM Histidine, 10 mM Phosphate, 4% Mannitol, 2% Trehalose,pH 7.0 with 0.1% Pluronic F68. Three different concentrations ofPEGylated hIFN with para-acetylphenylalanine substituted at position 35are used: 8 mg/ml, 12 mg/ml and 16 mg/ml. The study involves analyses att=0, 1 week, 8 weeks, and 24 weeks, and temperatures of 4° C., 25° C.,and 40° C. Methods involved in this study are as previously described:SDS-PAGE (reduced and non-reduced); SEC-HPLC, RP-HPLC, IEX, FTIR,moisture content, etc. Bioactivity is measured using a proliferationassay involving BrdU labeling. Briefly, this assay is performed withserum starved rat GHR (L43R) expressing BAF3 cell line, 2E2-2B12-F4.Cells are plated at specified densities of cells/well in a 96-wellplate. The cells are activated with a multi-point dose range ofPEGylated hGH polypeptide and are labeled at the same time with 50 uMBrdU (Sigma, St. Louis, Mo.). After 48 hours in culture, cells arefixed/permeabilized with 100 ul of BD cytofix/cytoperm solution (BDBiosciences) for 30 min at room temperature. To expose BrdU epitopes,fixed/permeablilized cells are treated with 30 ug/well of DNase (Sigma)for 1 hour at 37° C. Immunofluorescent staining with APC-conjugatedanti-BrdU antibody (BD Biosciences) enables sample analysis on the FACSArray. Variations to this method are known to those of ordinary skill inthe art.

TABLE 117 Time Buffer Conc. Point Temp. Analyses H7.3MT (30 mM  8 mg/mlzero SDS-PAGE (r + Nr), SEC, RP, Histidine, 4% cIEX, FTIR, moisturecontent, mannitol, 2% bioactivity Trehalose, pH 7.3) H7.3MT  8 mg/ml 1 w40 C. SDS-PAGE (r + Nr), SEC, RP, cIEX, bioactivity H7.3MT  8 mg/ml 8 w 4 C. SDS-PAGE (r + Nr), SEC, RP, cIEX H7.3MT  8 mg/ml 24 w   4 C.SDS-PAGE (r + Nr), SEC, RP, cIEX, FTIR, moisture content, bioactivityH7.3MT 12 mg/ml zero SDS-PAGE (r + Nr), SEC, RP, cIEX, FTIR, moisturecontent, bioactivity H7.3MT 12 mg/ml 1 w 40 C. SDS-PAGE (r + Nr), SEC,RP, cIEX, bioactivity H7.3MT 12 mg/ml 8 w  4 C. SDS-PAGE (r + Nr), SEC,RP, cIEX H7.3MT 12 mg/ml 8 w 25 C. SDS-PAGE (r + Nr), SEC, RP, cIEXH7.3MT 12 mg/ml 24 w   4 C. SDS-PAGE (r + Nr), SEC, RP, cIEX, FTIR,moisture content, bioactivity H7.3MT 16 mg/ml zero SDS-PAGE (r + Nr),SEC, RP, cIEX, FTIR, moisture content, bioactivity H7.3MT 16 mg/ml 1 w40 C. SDS-PAGE (r + Nr), SEC, RP, cIEX, bioactivity H7.3MT 16 mg/ml 8 w 4 C. SDS-PAGE (r + Nr), SEC, RP, cIEX H7.3MT 16 mg/ml 8 w 25 C.SDS-PAGE (r + Nr), SEC, RP, cIEX H7.3MT 16 mg/ml 24 w   4 C. SDS-PAGE(r + Nr), SEC, RP, cIEX, FTIR, moisture content, bioactivity H7.3MT+F  8mg/ml zero SDS-PAGE (r + Nr), SEC, RP, cIEX, FTIR, moisture content,bioactivity H7.3MT+F  8 mg/ml 1 w 40 C. SDS-PAGE (r + Nr), SEC, RP,cIEX, bioactivity H7.3MT+F  8 mg/ml 8 w  4 C. SDS-PAGE (r + Nr), SEC,RP, cIEX H7.3MT+F  8 mg/ml 24 w   4 C. SDS-PAGE (r + Nr), SEC, RP, cIEX,FTIR, moisture content, bioactivity H7.3MT+F 12 mg/ml zero SDS-PAGE (r +Nr), SEC, RP, cIEX, FTIR, moisture content, bioactivity H7.3MT+F 12mg/ml 1 w 40 C. SDS-PAGE (r + Nr), SEC, RP, cIEX, bioactivity H7.3MT+F12 mg/ml 8 w  4 C. SDS-PAGE (r + Nr), SEC, RP, cIEX H7.3MT+F 12 mg/ml 8w 25 C. SDS-PAGE (r + Nr), SEC, RP, cIEX H7.3MT+F 12 mg/ml 24 w   4 C.SDS-PAGE (r + Nr), SEC, RP, cIEX, FTIR, moisture content, bioactivityH7.3MT+F 16 mg/ml zero SDS-PAGE (r + Nr), SEC, RP, cIEX, FTIR, moisturecontent, bioactivity H7.3MT+F 16 mg/ml 1 w 40 C. SDS-PAGE (r + Nr), SEC,RP, cIEX, bioactivity H7.3MT+F 16 mg/ml 8 w  4 C. SDS-PAGE (r + Nr),SEC, RP, cIEX H7.3MT+F 16 mg/ml 8 w 25 C. SDS-PAGE (r + Nr), SEC, RP,cIEX H7.3MT+F 16 mg/ml 24 w   4 C. SDS-PAGE (r + Nr), SEC, RP, cIEX,FTIR, moisture content, bioactivity HP7MT (10 mM  8 mg/ml zero SDS-PAGE(r + Nr), SEC, RP, Histidine, 10 mM cIEX, FTIR, moisture content,Phosphate, 4% bioactivity mannitol, 2% Trehalose, pH 7.0) HP7MT  8 mg/ml1 w 40 C. SDS-PAGE (r + Nr), SEC, RP, cIEX, bioactivity HP7MT  8 mg/ml 8w  4 C. SDS-PAGE (r + Nr), SEC, RP, cIEX, HP7MT  8 mg/ml 24 w   4 C.SDS-PAGE (r + Nr), SEC, RP, cIEX, FTIR, moisture content, bioactivityHP7MT 12 mg/ml zero SDS-PAGE (r + Nr), SEC, RP, cIEX, FTIR, moisturecontent, bioactivity HP7MT 12 mg/ml 1 w 40 C. SDS-PAGE (r + Nr), SEC,RP, cIEX, bioactivity HP7MT 12 mg/ml 8 w  4 C. SDS-PAGE (r + Nr), SEC,RP, cIEX HP7MT 12 mg/ml 8 w 25 C. SDS-PAGE (r + Nr), SEC, RP, cIEX HP7MT12 mg/ml 24 w   4 C. SDS-PAGE (r + Nr), SEC, RP, cIEX, FTIR, moisturecontent, bioactivity HP7MT 16 mg/ml zero SDS-PAGE (r + Nr), SEC, RP,cIEX, FTIR, moisture content, bioactivity HP7MT 16 mg/ml 1 w 40 C.SDS-PAGE (r + Nr), SEC, RP, cIEX, bioactivity HP7MT 16 mg/ml 8 w  4 C.SDS-PAGE (r + Nr), SEC, RP, cIEX HP7MT 16 mg/ml 8 w 25 C. SDS-PAGE (r +Nr), SEC, RP, cIEX HP7MT 16 mg/ml 24 w   4 C. SDS-PAGE (r + Nr), SEC,RP, cIEX, FTIR, moisture content, bioactivity HP7MT+F  8 mg/ml zeroSDS-PAGE (r + Nr), SEC, RP, cIEX, FTIR, moisture content, bioactivityHP7MT+F  8 mg/ml 1 w 40 C. SDS-PAGE (r + Nr), SEC, RP, cIEX, bioactivityHP7MT+F  8 mg/ml 8 w  4 C. SDS-PAGE (r + Nr), SEC, RP, cIEX HP7MT+F  8mg/ml 24 w   4 C. SDS-PAGE (r + Nr), SEC, RP, cIEX, FTIR, moisturecontent, bioactivity HP7MT+F 12 mg/ml zero SDS-PAGE (r + Nr), SEC, RP,cIEX, FTIR, moisture content, bioactivity HP7MT+F 12 mg/ml 1 w 40 C.SDS-PAGE (r + Nr), SEC, RP, cIEX, bioactivity HP7MT+F 12 mg/ml 8 w  4 C.SDS-PAGE (r + Nr), SEC, RP, cIEX HP7MT+F 12 mg/ml 8 w 25 C. SDS-PAGE(r + Nr), SEC, RP, cIEX HP7MT+F 12 mg/ml 24 w   4 C. SDS-PAGE (r + Nr),SEC, RP, cIEX, FTIR, moisture content, bioactivity HP7MT+F 16 mg/ml zeroSDS-PAGE (r + Nr), SEC, RP, cIEX, FTIR, moisture content, bioactivityHP7MT+F 16 mg/ml 1 w 40 C. SDS-PAGE (r + Nr), SEC, RP, cIEX, bioactivityHP7MT+F 16 mg/ml 8 w  4 C. SDS-PAGE (r + Nr), SEC, RP, cIEX HP7MT+F 16mg/ml 8 w 25 C. SDS-PAGE (r + Nr), SEC, RP, cIEX HP7MT+F 16 mg/ml 24 w  4 C. SDS-PAGE (r + Nr), SEC, RP, cIEX, FTIR, moisture content,bioactivity

Reconstitution Study

Formulation ID H7.3MT is 30 mM Histidine, 4% Mannitol, 2% Trehalose, pH7.3. Formulation ID H7.3MT+F is 30 mM Histidine, 4% Mannitol, 2%Trehalose, pH 7.3 with 0.1% Pluronic F68. Formulation ID HP7MT is 10 mMHistidine, 10 mM Phosphate, 4% Mannitol, 2% Trehalose, pH 7.0.Formulation ID HP7MT+F is 10 mM Histidine, 10 mM Phosphate, 4% Mannitol,2% Trehalose, pH 7.0 with 0.1% Pluronic F68. After lyophilized samplesare held at 4° C. for 8 weeks, samples are reconstituted and held atroom temperature for t=0, 4, 8, and 24 hours and analyzed with methodsdescribed previously. See Table 118.

TABLE 118 Holding after Time Reconstitution Buffer Conc. Point Temp. (atRT) Analyses (2) H7.3MT  8 mg/ml 8 w 4 C. 4, 8, 24 hours SEC, RP,(SDS-PAGE (r + Nr), cIEX only for 24 hr) H7.3MT 12 mg/ml 8 w 4 C. 4, 8,24 hours SEC, RP, (SDS-PAGE (r + Nr), cIEX only for 24 hr) H7.3MT 16mg/ml 8 w 4 C. 4, 8, 24 hours SEC, RP, (SDS-PAGE (r + Nr), cIEX only for24 hr) H7.3MT+F  8 mg/ml 8 w 4 C. 4, 8, 24 hours SEC, RP, (SDS-PAGE (r +Nr), cIEX only for 24 hr) H7.3MT+F 12 mg/ml 8 w 4 C. 4, 8, 24 hours SEC,RP, (SDS-PAGE (r + Nr), cIEX only for 24 hr) H7.3MT+F 16 mg/ml 8 w 4 C.4, 8, 24 hours SEC, RP, (SDS-PAGE (r + Nr), cIEX only for 24 hr) HP7MT 8 mg/ml 8 w 4 C. 4, 8, 24 hours SEC, RP, (SDS-PAGE (r + Nr), cIEX onlyfor 24 hr) HP7MT 12 mg/ml 8 w 4 C. 4, 8, 24 hours SEC, RP, (SDS-PAGE(r + Nr), cIEX only for 24 hr) HP7MT 16 mg/ml 8 w 4 C. 4, 8, 24 hoursSEC, RP, (SDS-PAGE (r + Nr), cIEX only for 24 hr) HP7MT+F  8 mg/ml 8 w 4C. 4, 8, 24 hours SEC, RP, (SDS-PAGE (r + Nr), cIEX only for 24 hr)HP7MT+F 12 mg/ml 8 w 4 C. 4, 8, 24 hours SEC, RP, (SDS-PAGE (r + Nr),cIEX only for 24 hr) HP7MT+F 16 mg/ml 8 w 4 C. 4, 8, 24 hours SEC, RP,(SDS-PAGE (r + Nr), cIEX only for 24 hr)

Injection Feasibility Study

Injection feasibility is tested with 8, 12, 16, and 25 mg/ml ofPEGylated hGH polypeptide (Formulation ID H7.3MT). Unconjugated PEG(Formulation ID H7.3MT) is also analyzed at approximately 16 mg/ml and25 mg/ml, as well as a buffer control without polypeptide or PEG. Aninstron machine is used, and 4 lbs of force is used with 27 and 29 gaugeneedles.

Modifications to various conditions and/or parameters to the techniquesdescribed herein are known to those of ordinary skill in the art.Methods such as those described above or other methods known to those ofordinary skill in the art may be used in formulation studies. Potencystudies on samples may be performed with assays known to those ofordinary skill in the art.

Suitable formulations include, but are not limited to, H7MT-P withPluronic Acid; H7MGT-P with Pluronic Acid; H7MT-P; H7MGT-P; H6MT-P withPluronic Acid; H6MGT-P with Pluronic Acid; H6MT-P; H6MGT-P; HP7MT; HP7MTwith Pluronic Acid; H7.3MT; H7.3MT with Pluronic Acid. Suitableformulations may have a pH range of about 6 to about 7.3. Suitableformulations may have a pH range of about 5.5 to about 8. Suitableformulations may include histidine at about 5 to about 30 mM. Suitableformulations may optionally include mannitol at up to about 60 g/L.Suitable formulations may optionally include trehalose at up to about 50g/L. Suitable formulations may optionally include glycine at up to about60 g/L.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons of ordinary skill in theart and are to be included within the spirit and purview of thisapplication and scope of the appended claims. All publications, patents,and patent applications cited herein are hereby incorporated byreference herein in their entirety for all purposes.

TABLE 119 Sequences Cited. SEQ ID # Sequence Name 1 Full-length aminoacid sequence of hGH 2 The mature amino acid sequence of hGH (isoform 1)3 The 20-kDa hGH variant in which residues 32-46 of hGH are deleted

1-53. (canceled)
 54. A method of treating a patient having a disordermodulated by human growth hormone (hGH) with an effective amount of apharmaceutical formulation of an hGH polypeptide comprising one or morenon-naturally encoded amino acids wherein said formulation comprises: a)a buffer; b) at least one carrier, excipient, or stabilizer; c) and apharmaceutically effective quantity of human growth hormone. 55.(canceled)